Compositions for use in identification of bacteria

ABSTRACT

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/409,535, filed April 21, 2006, which is a continuation-in-part ofU.S. application Ser. No. 11/060,135, filed Feb. 17, 2005 which claimsthe benefit of priority to U.S. Provisional Application Ser. No.60/545,425 filed Feb. 18, 2004; U.S. Provisional Application Ser. No.60/559,754, filed Apr. 5, 2004; U.S. Provisional Application Ser. No.60/632,862, filed Dec. 3, 2004; U.S. Provisional Application Ser. No.60/639,068, filed Dec. 22, 2004; and U.S. Provisional Application Ser.No. 60/648,188, filed Jan. 28, 2005. U.S. application Ser. No.11/409,535 is a also continuation-in-part of U.S. application Ser. No.10/728,486, filed Dec. 5, 2003 now U.S. Pat. No. 7,718,354 which claimsthe benefit of priority to U.S. Provisional Application Ser. No.60/501,926, filed Sep. 11, 2003. U.S. application Ser. No. 11/409,535also claims the benefit of priority to: U.S. Provisional ApplicationSer. No. 60/674,118, filed Apr. 21, 2005; U.S. Provisional ApplicationSer. No. 60/705,631, filed Aug. 3, 2005; U.S. Provisional ApplicationSer. No. 60/732,539, filed Nov. 1, 2005; and U.S. ProvisionalApplication Ser. No. 60/773,124, filed Feb. 13, 2006. Each of theabove-referenced U.S. Applications is incorporated herein by referencein its entirety. Methods disclosed in U.S. application Ser. Nos.09/891,793, 10/156,608, 10/405,756, 10/418,514, 10/660,122, 10/660,996,10/660,997, 10/660,998, 10/728,486, 11/060,135, and 11/073,362, arecommonly owned and incorporated herein by reference in their entiretyfor any purpose.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support under CDCcontracts RO1 CI000099-01. The United States Government has certainrights in the invention.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledDIBIS0083USC3SEQ.txt, created on Mar. 6, 2007 which is 252 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions, kits and methods for rapididentification and quantification of bacteria by molecular mass and basecomposition analysis.

BACKGROUND OF THE INVENTION

A problem in determining the cause of a natural infectious outbreak or abioterrorist attack is the sheer variety of organisms that can causehuman disease. There are over 1400 organisms infectious to humans; manyof these have the potential to emerge suddenly in a natural epidemic orto be used in a malicious attack by bioterrorists (Taylor et al. Philos.Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This numberdoes not include numerous strain variants, bioengineered versions, orpathogens that infect plants or animals.

Much of the new technology being developed for detection of biologicalweapons incorporates a polymerase chain reaction (PCR) step based uponthe use of highly specific primers and probes designed to selectivelydetect certain pathogenic organisms. Although this approach isappropriate for the most obvious bioterrorist organisms, like smallpoxand anthrax, experience has shown that it is very difficult to predictwhich of hundreds of possible pathogenic organisms might be employed ina terrorist attack. Likewise, naturally emerging human disease that hascaused devastating consequence in public health has come from unexpectedfamilies of bacteria, viruses, fungi, or protozoa. Plants and animalsalso have their natural burden of infectious disease agents and thereare equally important biosafety and security concerns for agriculture.

A major conundrum in public health protection, biodefense, andagricultural safety and security is that these disciplines need to beable to rapidly identify and characterize infectious agents, while thereis no existing technology with the breadth of function to meet thisneed. Currently used methods for identification of bacteria rely uponculturing the bacterium to effect isolation from other organisms and toobtain sufficient quantities of nucleic acid followed by sequencing ofthe nucleic acid, both processes which are time and labor intensive.

Mass spectrometry provides detailed information about the moleculesbeing analyzed, including high mass accuracy. It is also a process thatcan be easily automated. DNA chips with specific probes can onlydetermine the presence or absence of specifically anticipated organisms.Because there are hundreds of thousands of species of benign bacteria,some very similar in sequence to threat organisms, even arrays with10,000 probes lack the breadth needed to identify a particular organism.

The present invention provides oligonucleotide primers and compositionsand kits containing the oligonucleotide primers, which define bacterialbioagent identifying amplicons and, upon amplification, producecorresponding amplification products whose molecular masses provide themeans to identify bacteria, for example, at and below the speciestaxonomic level.

SUMMARY OF THE INVENTION

The present invention provides compositions, kits and methods for rapididentification and quantification of bacteria by molecular mass and basecomposition analysis.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 456.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1261.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1261.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 288.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1269.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 288 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1269.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 698.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1420.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 698 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1420.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 217.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1167

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 217 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1167.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 399.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1041.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 399 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1041.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 430.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1321.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 430 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1321.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 174.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 853.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 174 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 853.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 172.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1360.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 172 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1360.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 456 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1261.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 456 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1261 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 288:1269, 698:1420, 217:1167, 399:1041,430:1321, 174:853, and 172:1360.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 681.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1022.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1022.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 315.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1379.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 315 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1379.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 346.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 955.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 346 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 955.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 504.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1409.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 504 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1409.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 323.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1068.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 323 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1068.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 479.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 938.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 479 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 938.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 681 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1022.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 681 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1022 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 315:1379, 346:955, 504:1409, 323:1068,479:938.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 583.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 923.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 583 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 923.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 454.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1418.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 454 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1418.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 250.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 902.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 250 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 902.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 384.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 878.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 384 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 878.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 694.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1215.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 694 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1215.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 194.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1173.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 194 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1173.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 375.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 890.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 375 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 890.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 656.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1224.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 656 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1224.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 618.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1157.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 618 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1157.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 302.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 852.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 302 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 852.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 199.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 889.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 199 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 889.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 596.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1169.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 596 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1169.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 150.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1242.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 150 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1242.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 166.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1069.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 166 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1069.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 166.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1168.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 166 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1168.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 583 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 923 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 454:1418, 250:902, 384:878, 694:1215,194:1173, 375:890, 656:1224, 618:1157, 302:852, 199:889, 596:1169,150:1242, 166:1069 and 166:1168.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 437.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1137.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 437 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1137.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 530.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 891.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 530 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 891.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 474.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 869.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 474 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 869.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 268.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1284.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 268 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1284.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 418.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1301.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 418 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1301.

An embodiment is an oligonucleotide primer pair comprising a forward anda reverse primer, each comprising between 13 and 35 linked nucleotidesin length, designed to generate an amplicon that is between about 45 andabout 200 linked nucleotides in length, wherein said forward primercomprises at least 80% complementarity to a first region withinnucleotides 1-286 of a reference sequence, said reference sequence beinga sequence extraction of coordinates 830671-831072 of Genbank gi number21281729 (ttccacgaaacagatgaagaaattaacaaaaaagcgcacgctattttcaaacatggaatgactccaattatttgtgttggtgaaacagacgaagagcgtgaaagtggtaaagctaacgatgttgtaggtgagcaagttaagaaagctgttgcaggtttatctgaagatcaacttaaatcagttgtaattgcttatgagccaatctgggcaatcggaactggtaaatcatcaacatctgaagatgcaaatgaaatgtgtgcatttgtacgtcaaactattgctgacttatcaagcaaagaagtatcagaagcaactcgtattcaatatggtggtagtgttaaacctaacaacattaaagaatacatggcacaaactgatattgatggggcattagtaggtggc (SEQID NO.: 1465)), and wherein said reverse primer comprises at least 80%complementarity to a second region within nucleotides 1-286 of saidreference sequence.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 318.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1300.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 318 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1300.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 440.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1076.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 440 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1076.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 219.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1013.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 219 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1013.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 437 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1137 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 530:891, 474:869, 268:1284, 418:1301,318:1300, 440:1076 and 219:1013.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 325.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1163.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 325 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1163.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 278.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1039.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 278 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1039.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 465.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1037.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 465 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1037.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 148.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1172.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 148 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1172.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 190.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1254.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 190 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1254.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 266.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1094.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 266 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1094.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 508.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1297.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 508 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1297.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 259.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1060.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 259 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1060.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 325 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1163 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 278:1039: 465:1037, 148:1172, 190:1254,266:1094, 508:1297 and 259:1060.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 376.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1265.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 376 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1265.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 267.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1341.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 267 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1341.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 705.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1056.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 705 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1056.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 710.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1259.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 710 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1259.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 374.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1111.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 374 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1111.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 545.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 978.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 545 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 978.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 249.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1095.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 249 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1095.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 195.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1376.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 195 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1376.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 311.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1014.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 311 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1014.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 365.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1052.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 365 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1052.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 527.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1071.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 527 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1071.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 490.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1182.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 490 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1182.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 376 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1265 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 267:1341, 705:1056, 710:1259, 374:1111,545:978, 249:1095, 195:1376, 311:1014, 365:1052, 527:1071 and 490:1182.

In some embodiments, either or both of the primers of a primer paircomposition contain at least one modified nucleobase such as5-propynyluracil or 5-propynylcytosine for example.

In some embodiments, either or both of the primers of the primer paircomprises at least one universal nucleobase such as inosine for example.

In some embodiments, either or both of the primers of the primer paircomprises at least one non-templated T residue on the 5′-end.

In some embodiments, either or both of the primers of the primer paircomprises at least one non-template tag.

In some embodiments, either or both of the primers of the primer paircomprises at least one molecular mass modifying tag.

In some embodiments, the present invention provides primers andcompositions comprising pairs of primers, and kits containing the same,and methods for use in identification of bacteria. The primers aredesigned to produce amplification products of DNA encoding genes thathave conserved and variable regions across different subgroups andgenotypes of bacteria.

Some embodiments are kits that contain one or more of the primer paircompositions. In some embodiments, each member of the one or more primerpairs of the kit is of a length of 14 to 35 nucleobases and has 70% to100% sequence identity with the corresponding member from any of theprimer pairs listed in Table 2.

Some embodiments of the kits contain at least one calibrationpolynucleotide for use in quantitiation of bacteria in a given sample,and also for use as a positive control for amplification.

Some embodiments of the kits contain at least one anion exchangefunctional group linked to a magnetic bead.

In some embodiments, the present invention also provides methods foridentification of bacteria. Nucleic acid from the bacterium is amplifiedusing the primers described above to obtain an amplification product.The molecular mass of the amplification product is measured. Optionally,the base composition of the amplification product is determined from themolecular mass. The molecular mass or base composition is compared witha plurality of molecular masses or base compositions of known analogousbacterial identifying amplicons, wherein a match between the molecularmass or base composition and a member of the plurality of molecularmasses or base compositions identifies the bacterium. In someembodiments, the molecular mass is measured by mass spectrometry in amodality such as electrospray ionization (ESI) time of flight (TOF) massspectrometry or ESI Fourier transform ion cyclotron resonance (FTICR)mass spectrometry, for example. Other mass spectrometry techniques canalso be used to measure the molecular mass of bacterial bioagentidentifying amplicons.

In some embodiments, the present invention is also directed to a methodfor determining the presence or absence of a bacterium in a sample.Nucleic acid from the sample is amplified using the compositiondescribed above to obtain an amplification product. The molecular massof the amplification product is determined. Optionally, the basecomposition of the amplification product is determined from themolecular mass. The molecular mass or base composition of theamplification product is compared with the known molecular masses orbase compositions of one or more known analogous bacterial bioagentidentifying amplicons, wherein a match between the molecular mass orbase composition of the amplification product and the molecular mass orbase composition of one or more known bacterial bioagent identifyingamplicons indicates the presence of the bacterium in the sample. In someembodiments, the molecular mass is measured by mass spectrometry.

In some embodiments, the present invention also provides methods fordetermination of the quantity of an unknown bacterium in a sample. Thesample is contacted with the composition described above and a knownquantity of a calibration polynucleotide comprising a calibrationsequence. Nucleic acid from the unknown bacterium in the sample isconcurrently amplified with the composition described above and nucleicacid from the calibration polynucleotide in the sample is concurrentlyamplified with the composition described above to obtain a firstamplification product comprising a bacterial bioagent identifyingamplicon and a second amplification product comprising a calibrationamplicon. The molecular masses and abundances for the bacterial bioagentidentifying amplicon and the calibration amplicon are determined. Thebacterial bioagent identifying amplicon is distinguished from thecalibration amplicon based on molecular mass and comparison of bacterialbioagent identifying amplicon abundance and calibration ampliconabundance indicates the quantity of bacterium in the sample. In someembodiments, the base composition of the bacterial bioagent identifyingamplicon is determined.

In some embodiments, the present invention provides methods fordetecting or quantifying bacteria by combining a nucleic acidamplification process with a mass determination process. In someembodiments, such methods identify or otherwise analyze the bacterium bycomparing mass information from an amplification product with acalibration or control product. Such methods can be carried out in ahighly multiplexed and/or parallel manner allowing for the analysis ofas many as 300 samples per 24 hours on a single mass measurementplatform. The accuracy of the mass determination methods in someembodiments of the present invention permits allows for the ability todiscriminate between different bacteria such as, for example, variousgenotypes and drug resistant strains of Staphylococcus aureus.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary of the invention, as well as the followingdetailed description of the invention, is better understood when read inconjunction with the accompanying drawings which are included by way ofexample and not by way of limitation.

FIG. 1: process diagram illustrating a representative primer pairselection process.

FIG. 2: process diagram illustrating an embodiment of the calibrationmethod.

FIG. 3: common pathogenic bacteria and primer pair coverage. The primerpair number in the upper right hand corner of each polygon indicatesthat the primer pair can produce a bioagent identifying amplicon for allspecies within that polygon.

FIG. 4: a representative 3D diagram of base composition (axes A, G andC) of bioagent identifying amplicons obtained with primer pair number 14(a precursor of primer pair number 348 which targets 16S rRNA). Thediagram indicates that the experimentally determined base compositionsof the clinical samples (labeled NHRC samples) closely match the basecompositions expected for Streptococcus pyogenes and are distinct fromthe expected base compositions of other organisms.

FIG. 5: a representative mass spectrum of amplification productsindicating the presence of bioagent identifying amplicons ofStreptococcus pyogenes, Neisseria meningitidis, and Haemophilusinfluenzae obtained from amplification of nucleic acid from a clinicalsample with primer pair number 349 which targets 23S rRNA.Experimentally determined molecular masses and base compositions for thesense strand of each amplification product are shown.

FIG. 6: a representative mass spectrum of amplification productsrepresenting a bioagent identifying amplicon of Streptococcus pyogenes,and a calibration amplicon obtained from amplification of nucleic acidfrom a clinical sample with primer pair number 356 which targets rplB.The experimentally determined molecular mass and base composition forthe sense strand of the Streptococcus pyogenes amplification product isshown.

FIG. 7: a representative mass spectrum of an amplified nucleic acidmixture which contained the Ames strain of Bacillus anthracis, a knownquantity of combination calibration polynucleotide (SEQ ID NO: 1464),and primer pair number 350 which targets the capC gene on the virulenceplasmid pX02 of Bacillus anthracis. Calibration amplicons produced inthe amplification reaction are visible in the mass spectrum as indicatedand abundance data (peak height) are used to calculate the quantity ofthe Ames strain of Bacillus anthracis.

DEFINITIONS

As used herein, the term “abundance” refers to an amount. The amount maybe described in terms of concentration which are common in molecularbiology such as “copy number,” “pfu or plate-forming unit” which arewell known to those with ordinary skill. Concentration may be relativeto a known standard or may be absolute.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” also comprises “sampletemplate.”

As used herein the term “amplification” refers to a special case ofnucleic acid replication involving template specificity. It is to becontrasted with non-specific template replication (i.e., replicationthat is template-dependent but not dependent on a specific template).Template specificity is here distinguished from fidelity of replication(i.e., synthesis of the proper polynucleotide sequence) and nucleotide(ribo- or deoxyribo-) specificity. Template specificity is frequentlydescribed in terms of “target” specificity. Target sequences are“targets” in the sense that they are sought to be sorted out from othernucleic acid. Amplification techniques have been designed primarily forthis sorting out. Template specificity is achieved in most amplificationtechniques by the choice of enzyme. Amplification enzymes are enzymesthat, under conditions they are used, will process only specificsequences of nucleic acid in a heterogeneous mixture of nucleic acid.For example, in the case of Qβ replicase, MDV-1 RNA is the specifictemplate for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci.USA 69:3038 [1972]). Other nucleic acid will not be replicated by thisamplification enzyme. Similarly, in the case of T7 RNA polymerase, thisamplification enzyme has a stringent specificity for its own promoters(Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNAligase, the enzyme will not ligate the two oligonucleotides orpolynucleotides, where there is a mismatch between the oligonucleotideor polynucleotide substrate and the template at the ligation junction(D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq andPfu polymerases, by virtue of their ability to function at hightemperature, are found to display high specificity for the sequencesbounded and thus defined by the primers; the high temperature results inthermodynamic conditions that favor primer hybridization with the targetsequences and not hybridization with non-target sequences (H. A. Erlich(ed.), PCR Technology, Stockton Press [1989]).

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification, excluding primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “analogous” when used in context of comparisonof bioagent identifying amplicons indicates that the bioagentidentifying amplicons being compared are produced with the same pair ofprimers. For example, bioagent identifying amplicon “A” and bioagentidentifying amplicon “B”, produced with the same pair of primers areanalogous with respect to each other. Bioagent identifying amplicon “C”,produced with a different pair of primers is not analogous to eitherbioagent identifying amplicon “A” or bioagent identifying amplicon “B”.

As used herein, the term “anion exchange functional group” refers to apositively charged functional group capable of binding an anion throughan electrostatic interaction. The most well known anion exchangefunctional groups are the amines, including primary, secondary, tertiaryand quaternary amines.

The term “bacteria” or “bacterium” refers to any member of the groups ofeubacteria and archaebacteria.

As used herein, a “base composition” is the exact number of eachnucleobase (for example, A, T, C and G) in a segment of nucleic acid.For example, amplification of nucleic acid of Staphylococcus aureusstrain carrying the lukS-PV gene with primer pair number 2095 (SEQ IDNOs: 456:1261) produces an amplification product 117 nucleobases inlength from nucleic acid of the lukS-PV gene that has a base compositionof A35 G17 C19 T46 (by convention—with reference to the sense strand ofthe amplification product). Because the molecular masses of each of thefour natural nucleotides and chemical modifications thereof are known(if applicable), a measured molecular mass can be deconvoluted to a listof possible base compositions. Identification of a base composition of asense strand which is complementary to the corresponding antisensestrand in terms of base composition provides a confirmation of the truebase composition of an unknown amplification product. For example, thebase composition of the antisense strand of the 139 nucleobaseamplification product described above is A46 G19 C17 T35.

As used herein, a “base composition probability cloud” is arepresentation of the diversity in base composition resulting from avariation in sequence that occurs among different isolates of a givenspecies. The “base composition probability cloud” represents the basecomposition constraints for each species and is typically visualizedusing a pseudo four-dimensional plot.

In the context of this invention, a “bioagent” is any organism, cell, orvirus, living or dead, or a nucleic acid derived from such an organism,cell or virus. Examples of bioagents include, but are not limited, tocells, (including but not limited to human clinical samples, bacterialcells and other pathogens), viruses, fungi, protists, parasites, andpathogenicity markers (including but not limited to: pathogenicityislands, antibiotic resistance genes, virulence factors, toxin genes andother bioregulating compounds). Samples may be alive or dead or in avegetative state (for example, vegetative bacteria or spores) and may beencapsulated or bioengineered. In the context of this invention, a“pathogen” is a bioagent which causes a disease or disorder.

As used herein, a “bioagent division” is defined as group of bioagentsabove the species level and includes but is not limited to, orders,families, classes, clades, genera or other such groupings of bioagentsabove the species level.

As used herein, the term “bioagent identifying amplicon” refers to apolynucleotide that is amplified from a bioagent in an amplificationreaction and which 1) provides sufficient variability to distinguishamong bioagents from whose nucleic acid the bioagent identifyingamplicon is produced and 2) whose molecular mass is amenable to a rapidand convenient molecular mass determination modality such as massspectrometry, for example.

As used herein, the term “biological product” refers to any productoriginating from an organism. Biological products are often products ofprocesses of biotechnology. Examples of biological products include, butare not limited to: cultured cell lines, cellular components,antibodies, proteins and other cell-derived biomolecules, growth media,growth harvest fluids, natural products and bio-pharmaceutical products.

The terms “biowarfare agent” and “bioweapon” are synonymous and refer toa bacterium, virus, fungus or protozoan that could be deployed as aweapon to cause bodily harm to individuals. Military or terrorist groupsmay be implicated in deployment of biowarfare agents.

In context of this invention, the term “broad range survey primer pair”refers to a primer pair designed to produce bioagent identifyingamplicons across different broad groupings of bioagents. For example,the ribosomal RNA-targeted primer pairs are broad range survey primerpairs which have the capability of producing bacterial bioagentidentifying amplicons for essentially all known bacteria. With respectto broad range primer pairs employed for identification of bacteria, abroad range survey primer pair for bacteria such as 16S rRNA primer pairnumber 346 (SEQ ID NOs: 202:1110) for example, will produce an bacterialbioagent identifying amplicon for essentially all known bacteria.

The term “calibration amplicon” refers to a nucleic acid segmentrepresenting an amplification product obtained by amplification of acalibration sequence with a pair of primers designed to produce abioagent identifying amplicon.

The term “calibration sequence” refers to a polynucleotide sequence towhich a given pair of primers hybridizes for the purpose of producing aninternal (i.e.: included in the reaction) calibration standardamplification product for use in determining the quantity of a bioagentin a sample. The calibration sequence may be expressly added to anamplification reaction, or may already be present in the sample prior toanalysis.

The term “clade primer pair” refers to a primer pair designed to producebioagent identifying amplicons for species belonging to a clade group. Aclade primer pair may also be considered as a “speciating” primer pairwhich is useful for distinguishing among closely related species.

The term “codon” refers to a set of three adjoined nucleotides (triplet)that codes for an amino acid or a termination signal.

In context of this invention, the term “codon base compositionanalysis,” refers to determination of the base composition of anindividual codon by obtaining a bioagent identifying amplicon thatincludes the codon. The bioagent identifying amplicon will at leastinclude regions of the target nucleic acid sequence to which the primershybridize for generation of the bioagent identifying amplicon as well asthe codon being analyzed, located between the two primer hybridizationregions.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. For example, for the sequence “5′-A-G-T-3′,” is complementary tothe sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in whichonly some of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids. Either term may also be usedin reference to individual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid strand, in contrast orcomparison to the complementarity between the rest of theoligonucleotide and the nucleic acid strand.

The term “complement of a nucleic acid sequence” as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.Where a first oligonucleotide is complementary to a region of a targetnucleic acid and a second oligonucleotide has complementary to the sameregion (or a portion of this region) a “region of overlap” exists alongthe target nucleic acid. The degree of overlap will vary depending uponthe extent of the complementarity.

In context of this invention, the term “division-wide primer pair”refers to a primer pair designed to produce bioagent identifyingamplicons within sections of a broader spectrum of bioagents Forexample, primer pair number 352 (SEQ ID NOs: 687:1411), a division-wideprimer pair, is designed to produce bacterial bioagent identifyingamplicons for members of the Bacillus group of bacteria which comprises,for example, members of the genera Streptococci, Enterococci, andStaphylococci. Other division-wide primer pairs may be used to producebacterial bioagent identifying amplicons for other groups of bacterialbioagents.

As used herein, the term “concurrently amplifying” used with respect tomore than one amplification reaction refers to the act of simultaneouslyamplifying more than one nucleic acid in a single reaction mixture.

As used herein, the term “drill-down primer pair” refers to a primerpair designed to produce bioagent identifying amplicons foridentification of sub-species characteristics or confirmation of aspecies assignment. For example, primer pair number 2146 (SEQ ID NOs:437:1137), a drill-down Staphylococcus aureus genotyping primer pair, isdesigned to produce Staphylococcus aureus genotyping amplicons. Otherdrill-down primer pairs may be used to produce bioagent identifyingamplicons for Staphylococcus aureus and other bacterial species.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding the their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

As used herein, the term “etiology” refers to the causes or origins, ofdiseases or abnormal physiological conditions.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

The terms “homology,” “homologous” and “sequence identity” refer to adegree of identity. There may be partial homology or complete homology.A partially homologous sequence is one that is less than 100% identicalto another sequence. Determination of sequence identity is described inthe following example: a primer 20 nucleobases in length which isotherwise identical to another 20 nucleobase primer but having twonon-identical residues has 18 of 20 identical residues (18/20=0.9 or 90%sequence identity). In another example, a primer 15 nucleobases inlength having all residues identical to a 15 nucleobase segment of aprimer 20 nucleobases in length would have 15/20=0.75 or 75% sequenceidentity with the 20 nucleobase primer. In context of the presentinvention, sequence identity is meant to be properly determined when thequery sequence and the subject sequence are both described and alignedin the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST,will return results in two different alignment orientations. In thePlus/Plus orientation, both the query sequence and the subject sequenceare aligned in the 5′ to 3′ direction. On the other hand, in thePlus/Minus orientation, the query sequence is in the 5′ to 3′ directionwhile the subject sequence is in the 3′ to 5′ direction. It should beunderstood that with respect to the primers of the present invention,sequence identity is properly determined when the alignment isdesignated as Plus/Plus. Sequence identity may also encompass alternateor modified nucleobases that perform in a functionally similar manner tothe regular nucleobases adenine, thymine, guanine and cytosine withrespect to hybridization and primer extension in amplificationreactions. In a non-limiting example, if the 5-propynyl pyrimidinespropyne C and/or propyne T replace one or more C or T residues in oneprimer which is otherwise identical to another primer in sequence andlength, the two primers will have 100% sequence identity with eachother. In another non-limiting example, Inosine (I) may be used as areplacement for G or T and effectively hybridize to C, A or U (uracil).Thus, if inosine replaces one or more C, A or U residues in one primerwhich is otherwise identical to another primer in sequence and length,the two primers will have 100% sequence identity with each other. Othersuch modified or universal bases may exist which would perform in afunctionally similar manner for hybridization and amplificationreactions and will be understood to fall within this definition ofsequence identity.

As used herein, “housekeeping gene” refers to a gene encoding a proteinor RNA involved in basic functions required for survival andreproduction of a bioagent. Housekeeping genes include, but are notlimited to genes encoding RNA or proteins involved in translation,replication, recombination and repair, transcription, nucleotidemetabolism, amino acid metabolism, lipid metabolism, energy generation,uptake, secretion and the like.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology.

The term “in silico” refers to processes taking place via computercalculations. For example, electronic PCR (ePCR) is a process analogousto ordinary PCR except that it is carried out using nucleic acidsequences and primer pair sequences stored on a computer formattedmedium.

As used herein, “intelligent primers” are primers that are designed tobind to highly conserved sequence regions of a bioagent identifyingamplicon that flank an intervening variable region and, uponamplification, yield amplification products which ideally provide enoughvariability to distinguish individual bioagents, and which are amenableto molecular mass analysis. By the term “highly conserved,” it is meantthat the sequence regions exhibit between about 80-100%, or betweenabout 90-100%, or between about 95-100% identity among all, or at least70%, at least 80%, at least 90%, at least 95%, or at least 99% ofspecies or strains.

The “ligase chain reaction” (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, that hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. However, because the four oligonucleotides used inthis assay can pair to form two short ligatable fragments, there is thepotential for the generation of target-independent background signal.The use of LCR for mutant screening is limited to the examination ofspecific nucleic acid positions.

The term “locked nucleic acid” or “LNA” refers to a nucleic acidanalogue containing one or more 2′-O, 4′-C-methylene-β-D-ribofuranosylnucleotide monomers in an RNA mimicking sugar conformation. LNAoligonucleotides display unprecedented hybridization affinity towardcomplementary single-stranded RNA and complementary single- ordouble-stranded DNA. LNA oligonucleotides induce A-type (RNA-like)duplex conformations. The primers of the present invention may containLNA modifications.

As used herein, the term “mass-modifying tag” refers to any modificationto a given nucleotide which results in an increase in mass relative tothe analogous non-mass modified nucleotide. Mass-modifying tags caninclude heavy isotopes of one or more elements included in thenucleotide such as carbon-13 for example. Other possible modificationsinclude addition of substituents such as iodine or bromine at the 5position of the nucleobase for example.

The term “mass spectrometry” refers to measurement of the mass of atomsor molecules. The molecules are first converted to ions, which areseparated using electric or magnetic fields according to the ratio oftheir mass to electric charge. The measured masses are used to identitythe molecules.

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi; and ciliates.

The term “multi-drug resistant” or multiple-drug resistant”refers to amicroorganism which is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

The term “multiplex PCR” refers to a PCR reaction where more than oneprimer set is included in the reaction pool allowing 2 or more differentDNA targets to be amplified by PCR in a single reaction tube.

The term “non-template tag” refers to a stretch of at least threeguanine or cytosine nucleobases of a primer used to produce a bioagentidentifying amplicon which are not complementary to the template. Anon-template tag is incorporated into a primer for the purpose ofincreasing the primer-duplex stability of later cycles of amplificationby incorporation of extra G-C pairs which each have one additionalhydrogen bond relative to an A-T pair.

The term “nucleic acid sequence” as used herein refers to the linearcomposition of the nucleic acid residues A, T, C or G or anymodifications thereof, within an oligonucleotide, nucleotide orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be single or double stranded, andrepresent the sense or antisense strand

As used herein, the term “nucleobase” is synonymous with other terms inuse in the art including “nucleotide,” “deoxynucleotide,” “nucleotideresidue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP).

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 5-propynyl pyrimidines(i.e., 5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 13 to35 nucleotides. The exact size will depend on many factors, which inturn depend on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, PCR, or a combinationthereof. Because mononucleotides are reacted to make oligonucleotides ina manner such that the 5′ phosphate of one mononucleotide pentose ringis attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′-end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′-end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction. Alloligonucleotide primers disclosed herein are understood to be presentedin the 5′ to 3′ direction when reading left to right. When twodifferent, non-overlapping oligonucleotides anneal to different regionsof the same linear complementary nucleic acid sequence, and the 3′ endof one oligonucleotide points towards the 5′ end of the other, theformer may be called the “upstream” oligonucleotide and the latter the“downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

In the context of this invention, a “pathogen” is a bioagent whichcauses a disease or disorder.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

The term “peptide nucleic acid” (“PNA”) as used herein refers to amolecule comprising bases or base analogs such as would be found innatural nucleic acid, but attached to a peptide backbone rather than thesugar-phosphate backbone typical of nucleic acids. The attachment of thebases to the peptide is such as to allow the bases to base pair withcomplementary bases of nucleic acid in a manner similar to that of anoligonucleotide. These small molecules, also designated anti geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63).The primers of the present invention may comprise PNAs.

The term “polymerase” refers to an enzyme having the ability tosynthesize a complementary strand of nucleic acid from a startingtemplate nucleic acid strand and free dNTPs.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference, that describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.” With PCR, it is possible to amplify a single copy ofa specific target sequence in genomic DNA to a level detectable byseveral different methodologies (e.g., hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.

The term “polymerization means” or “polymerization agent” refers to anyagent capable of facilitating the addition of nucleoside triphosphatesto an oligonucleotide. Preferred polymerization means comprise DNA andRNA polymerases.

As used herein, the terms “pair of primers,” or “primer pair” aresynonymous. A primer pair is used for amplification of a nucleic acidsequence. A pair of primers comprises a forward primer and a reverseprimer. The forward primer hybridizes to a sense strand of a target genesequence to be amplified and primes synthesis of an antisense strand(complementary to the sense strand) using the target sequence as atemplate. A reverse primer hybridizes to the antisense strand of atarget gene sequence to be amplified and primes synthesis of a sensestrand (complementary to the antisense strand) using the target sequenceas a template.

The primers are designed to bind to highly conserved sequence regions ofa bioagent identifying amplicon that flank an intervening variableregion and yield amplification products which ideally provide enoughvariability to distinguish each individual bioagent, and which areamenable to molecular mass analysis. In some embodiments, the highlyconserved sequence regions exhibit between about 80-100%, or betweenabout 90-100%, or between about 95-100% identity, or between about99-100% identity. The molecular mass of a given amplification productprovides a means of identifying the bioagent from which it was obtained,due to the variability of the variable region. Thus design of theprimers requires selection of a variable region with appropriatevariability to resolve the identity of a given bioagent. Bioagentidentifying amplicons are ideally specific to the identity of thebioagent.

Properties of the primers may include any number of properties relatedto structure including, but not limited to: nucleobase length which maybe contiguous (linked together) or non-contiguous (for example, two ormore contiguous segments which are joined by a linker or loop moiety),modified or universal nucleobases (used for specific purposes such asfor example, increasing hybridization affinity, preventing non-templatedadenylation and modifying molecular mass) percent complementarity to agiven target sequences.

Properties of the primers also include functional features including,but not limited to, orientation of hybridization (forward or reverse)relative to a nucleic acid template. The coding or sense strand is thestrand to which the forward priming primer hybridizes (forward primingorientation) while the reverse priming primer hybridizes to thenon-coding or antisense strand (reverse priming orientation). Thefunctional properties of a given primer pair also include the generictemplate nucleic acid to which the primer pair hybridizes. For example,identification of bioagents can be accomplished at different levelsusing primers suited to resolution of each individual level ofidentification. Broad range survey primers are designed with theobjective of identifying a bioagent as a member of a particular division(e.g., an order, family, genus or other such grouping of bioagents abovethe species level of bioagents). In some embodiments, broad range surveyintelligent primers are capable of identification of bioagents at thespecies or sub-species level. Other primers may have the functionalityof producing bioagent identifying amplicons for members of a giventaxonomic genus, clade, species, sub-species or genotype (includinggenetic variants which may include presence of virulence genes orantibiotic resistance genes or mutations). Additional functionalproperties of primer pairs include the functionality of performingamplification either singly (single primer pair per amplificationreaction vessel) or in a multiplex fashion (multiple primer pairs andmultiple amplification reactions within a single reaction vessel).

As used herein, the terms “purified” or “substantially purified” referto molecules, either nucleic or amino acid sequences, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

The term “reverse transcriptase” refers to an enzyme having the abilityto transcribe DNA from an RNA template. This enzymatic activity is knownas reverse transcriptase activity. Reverse transcriptase activity isdesirable in order to obtain DNA from RNA viruses which can then beamplified and analyzed by the methods of the present invention.

The term “ribosomal RNA” or “rRNA” refers to the primary ribonucleicacid constituent of ribosomes. Ribosomes are the protein-manufacturingorganelles of cells and exist in the cytoplasm. Ribosomal RNAs aretranscribed from the DNA genes encoding them.

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin. Biological samples may beanimal, including human, fluid, solid (e.g., stool) or tissue, as wellas liquid and solid food and feed products and ingredients such as dairyitems, vegetables, meat and meat by-products, and waste. Biologicalsamples may be obtained from all of the various families of domesticanimals, as well as feral or wild animals, including, but not limitedto, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.Environmental samples include environmental material such as surfacematter, soil, water, air and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention. The term “source of target nucleic acid” refers toany sample that contains nucleic acids (RNA or DNA). Particularlypreferred sources of target nucleic acids are biological samplesincluding, but not limited to blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum and semen.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is often a contaminant. It maybe the result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified away from the sample. Forexample, nucleic acids from organisms other than those to be detectedmay be present as background in a test sample.

A “segment” is defined herein as a region of nucleic acid within atarget sequence.

The “self-sustained sequence replication reaction” (3SR) (Guatelli etal., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum atProc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based invitro amplification system (Kwok et al., Proc. Natl. Acad. Sci.,86:1173-1177 [1989]) that can exponentially amplify RNA sequences at auniform temperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

As used herein, the term “sequence alignment” refers to a listing ofmultiple DNA or amino acid sequences and aligns them to highlight theirsimilarities. The listings can be made using bioinformatics computerprograms.

In context of this invention, the term “speciating primer pair” refersto a primer pair designed to produce a bioagent identifying ampliconwith the diagnostic capability of identifying species members of a groupof genera or a particular genus of bioagents. Primer pair number 2249(SEQ ID NOs: 430:1321), for example, is a speciating primer pair used todistinguish Staphylococcus aureus from other species of the genusStaphylococcus.

As used herein, a “sub-species characteristic” is a geneticcharacteristic that provides the means to distinguish two members of thesame bioagent species. For example, one viral strain could bedistinguished from another viral strain of the same species bypossessing a genetic change (e.g., for example, a nucleotide deletion,addition or substitution) in one of the viral genes, such as theRNA-dependent RNA polymerase. Sub-species characteristics such asvirulence genes and drug-are responsible for the phenotypic differencesamong the different strains of bacteria.

As used herein, the term “target” is used in a broad sense to indicatethe gene or genomic region being amplified by the primers. Because thepresent invention provides a plurality of amplification products fromany given primer pair (depending on the bioagent being analyzed),multiple amplification products from different specific nucleic acidsequences may be obtained. Thus, the term “target” is not used to referto a single specific nucleic acid sequence. The “target” is sought to besorted out from other nucleic acid sequences and contains a sequencethat has at least partial complementarity with an oligonucleotideprimer. The target nucleic acid may comprise single- or double-strandedDNA or RNA. A “segment” is defined as a region of nucleic acid withinthe target sequence.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr.Thermodynamics and NMR of internal G. T mismatches in DNA. Biochemistry36, 10581-94 (1997) include more sophisticated computations which takestructural and environmental, as well as sequence characteristics intoaccount for the calculation of T_(m).

The term “triangulation genotyping analysis” refers to a method ofgenotyping a bioagent by measurement of molecular masses or basecompositions of amplification products, corresponding to bioagentidentifying amplicons, obtained by amplification of regions of more thanone gene. In this sense, the term “triangulation” refers to a method ofestablishing the accuracy of information by comparing three or moretypes of independent points of view bearing on the same findings.Triangulation genotyping analysis carried out with a plurality oftriangulation genotyping analysis primers yields a plurality of basecompositions that then provide a pattern or “barcode” from which aspecies type can be assigned. The species type may represent apreviously known sub-species or strain, or may be a previously unknownstrain having a specific and previously unobserved base compositionbarcode indicating the existence of a previously unknown genotype.

As used herein, the term “triangulation genotyping analysis primer pair”is a primer pair designed to produce bioagent identifying amplicons fordetermining species types in a triangulation genotyping analysis.

The employment of more than one bioagent identifying amplicon foridentification of a bioagent is herein referred to as “triangulationidentification.” Triangulation identification is pursued by analyzing aplurality of bioagent identifying amplicons produced with differentprimer pairs. This process is used to reduce false negative and falsepositive signals, and enable reconstruction of the origin of hybrid orotherwise engineered bioagents. For example, identification of the threepart toxin genes typical of B. anthracis (Bowen et al., J. Appl.Microbiol., 1999, 87, 270-278) in the absence of the expected signaturesfrom the B. anthracis genome would suggest a genetic engineering event.

In the context of this invention, the term “unknown bioagent” may meaneither: (i) a bioagent whose existence is known (such as the well knownbacterial species Staphylococcus aureus for example) but which is notknown to be in a sample to be analyzed, or (ii) a bioagent whoseexistence is not known (for example, the SARS coronavirus was unknownprior to April 2003). For example, if the method for identification ofcoronaviruses disclosed in commonly owned U.S. patent Ser. No.10/829,826 (incorporated herein by reference in its entirety) was to beemployed prior to April 2003 to identify the SARS coronavirus in aclinical sample, both meanings of “unknown” bioagent are applicablesince the SARS coronavirus was unknown to science prior to April, 2003and since it was not known what bioagent (in this case a coronavirus)was present in the sample. On the other hand, if the method of U.S.patent Ser. No. 10/829,826 was to be employed subsequent to April 2003to identify the SARS coronavirus in a clinical sample, only the firstmeaning (i) of “unknown” bioagent would apply since the SARS coronavirusbecame known to science subsequent to April 2003 and since it was notknown what bioagent was present in the sample.

The term “variable sequence” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, the genesof two different bacterial species may vary in sequence by the presenceof single base substitutions and/or deletions or insertions of one ormore nucleotides. These two forms of the structural gene are said tovary in sequence from one another. In the context of the presentinvention, “viral nucleic acid” includes, but is not limited to, DNA,RNA, or DNA that has been obtained from viral RNA, such as, for example,by performing a reverse transcription reaction. Viral RNA can either besingle-stranded (of positive or negative polarity) or double-stranded.

The term “virus” refers to obligate, ultramicroscopic, parasites thatare incapable of autonomous replication (i.e., replication requires theuse of the host cell's machinery). Viruses can survive outside of a hostcell but cannot replicate.

The term “wild-type” refers to a gene or a gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified”, “mutant” or “polymorphic” refers to a gene or gene productthat displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

As used herein, a “wobble base” is a variation in a codon found at thethird nucleotide position of a DNA triplet. Variations in conservedregions of sequence are often found at the third nucleotide position dueto redundancy in the amino acid code.

DETAILED DESCRIPTION OF EMBODIMENTS

A. Bioagent Identifying Amplicons

The present invention provides methods for detection and identificationof unknown bioagents using bioagent identifying amplicons. Primers areselected to hybridize to conserved sequence regions of nucleic acidsderived from a bioagent, and which bracket variable sequence regions toyield a bioagent identifying amplicon, which can be amplified and whichis amenable to molecular mass determination. The molecular mass thenprovides a means to uniquely identify the bioagent without a requirementfor prior knowledge of the possible identity of the bioagent. Themolecular mass or corresponding base composition signature of theamplification product is then matched against a database of molecularmasses or base composition signatures. A match is obtained when anexperimentally-determined molecular mass or base composition of ananalyzed amplification product is compared with known molecular massesor base compositions of known bioagent identifying amplicons and theexperimentally determined molecular mass or base composition is the sameas the molecular mass or base composition of one of the known bioagentidentifying amplicons. Alternatively, the experimentally-determinedmolecular mass or base composition may be within experimental error ofthe molecular mass or base composition of a known bioagent identifyingamplicon and still be classified as a match. In some cases, the matchmay also be classified using a probability of match model such as themodels described in U.S. Ser. No. 11/073,362, which is commonly ownedand incorporated herein by reference in entirety. Furthermore, themethod can be applied to rapid parallel multiplex analyses, the resultsof which can be employed in a triangulation identification strategy. Thepresent method provides rapid throughput and does not require nucleicacid sequencing of the amplified target sequence for bioagent detectionand identification.

Despite enormous biological diversity, all forms of life on earth sharesets of essential, common features in their genomes. Since genetic dataprovide the underlying basis for identification of bioagents by themethods of the present invention, it is necessary to select segments ofnucleic acids which ideally provide enough variability to distinguisheach individual bioagent and whose molecular mass is amenable tomolecular mass determination.

Unlike bacterial genomes, which exhibit conservation of numerous genes(i.e. housekeeping genes) across all organisms, viruses do not share agene that is essential and conserved among all virus families.Therefore, viral identification is achieved within smaller groups ofrelated viruses, such as members of a particular virus family or genus.For example, RNA-dependent RNA polymerase is present in allsingle-stranded RNA viruses and can be used for broad priming as well asresolution within the virus family.

In some embodiments of the present invention, at least one bacterialnucleic acid segment is amplified in the process of identifying thebacterial bioagent. Thus, the nucleic acid segments that can beamplified by the primers disclosed herein and that provide enoughvariability to distinguish each individual bioagent and whose molecularmasses are amenable to molecular mass determination are herein describedas bioagent identifying amplicons.

In some embodiments of the present invention, bioagent identifyingamplicons comprise from about 45 to about 150 nucleobases (i.e. fromabout 45 to about 200 linked nucleosides), although both longer andshort regions may be used. One of ordinary skill in the art willappreciate that the invention embodies compounds of 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, and 150 nucleobases in length, or any rangetherewithin.

It is the combination of the portions of the bioagent nucleic acidsegment to which the primers hybridize (hybridization sites) and thevariable region between the primer hybridization sites that comprisesthe bioagent identifying amplicon. Thus, it can be said that a givenbioagent identifying amplicon is “defined by” a given pair of primers.

In some embodiments, bioagent identifying amplicons amenable tomolecular mass determination which are produced by the primers describedherein are either of a length, size or mass compatible with theparticular mode of molecular mass determination or compatible with ameans of providing a predictable fragmentation pattern in order toobtain predictable fragments of a length compatible with the particularmode of molecular mass determination. Such means of providing apredictable fragmentation pattern of an amplification product include,but are not limited to, cleavage with chemical reagents, restrictionenzymes or cleavage primers, for example. Thus, in some embodiments,bioagent identifying amplicons are larger than 150 nucleobases and areamenable to molecular mass determination following restrictiondigestion. Methods of using restriction enzymes and cleavage primers arewell known to those with ordinary skill in the art.

In some embodiments, amplification products corresponding to bioagentidentifying amplicons are obtained using the polymerase chain reaction(PCR) that is a routine method to those with ordinary skill in themolecular biology arts. Other amplification methods may be used such asligase chain reaction (LCR), low-stringency single primer PCR, andmultiple strand displacement amplification (MDA). These methods are alsoknown to those with ordinary skill.

B. Primers and Primer Pairs

In some embodiments, the primers are designed to bind to conservedsequence regions of a bioagent identifying amplicon that flank anintervening variable region and yield amplification products whichprovide variability sufficient to distinguish each individual bioagent,and which are amenable to molecular mass analysis. In some embodiments,the highly conserved sequence regions exhibit between about 80-100%, orbetween about 90-100%, or between about 95-100% identity, or betweenabout 99-100% identity. The molecular mass of a given amplificationproduct provides a means of identifying the bioagent from which it wasobtained, due to the variability of the variable region. Thus, design ofthe primers involves selection of a variable region with sufficientvariability to resolve the identity of a given bioagent. In someembodiments, bioagent identifying amplicons are specific to the identityof the bioagent.

In some embodiments, identification of bioagents is accomplished atdifferent levels using primers suited to resolution of each individuallevel of identification. Broad range survey primers are designed withthe objective of identifying a bioagent as a member of a particulardivision (e.g., an order, family, genus or other such grouping ofbioagents above the species level of bioagents). In some embodiments,broad range survey intelligent primers are capable of identification ofbioagents at the species or sub-species level. Examples of broad rangesurvey primers include, but are not limited to: primer pair numbers: 346(SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 SEQ ID NOs:706:895), and 361 (SEQ ID NOs: 697:1398) which target DNA encoding 16SrRNA, and primer pair numbers 349 (SEQ ID NOs: 401:1156) and 360 (SEQ IDNOs: 409:1434) which target DNA encoding 23S rRNA.

In some embodiments, drill-down primers are designed with the objectiveof identifying a bioagent at the sub-species level (including strains,subtypes, variants and isolates) based on sub-species characteristicswhich may, for example, include single nucleotide polymorphisms (SNPs),variable number tandem repeats (VNTRs), deletions, drug resistancemutations or any other modification of a nucleic acid sequence of abioagent relative to other members of a species having differentsub-species characteristics. Drill-down intelligent primers are notalways required for identification at the sub-species level becausebroad range survey intelligent primers may, in some cases providesufficient identification resolution to accomplishing thisidentification objective. Examples of drill-down primers include, butare not limited to: confirmation primer pairs such as primer pairnumbers 351 (SEQ ID NOs: 355:1423) and 353 (SEQ ID NOs: 220:1394), whichtarget the pX01 virulence plasmid of Bacillus anthracis. Other examplesof drill-down primer pairs are found in sets of triangulation genotypingprimer pairs such as, for example, the primer pair number 2146 (SEQ IDNOs: 437:1137) which targets the arcC gene (encoding carmabate kinase)and is included in an 8 primer pair panel or kit for use in genotypingStaphylococcus aureus, or in other panels or kits of primer pairs usedfor determining drug-resistant bacterial strains, such as, for example,primer pair number 2095 (SEQ ID NOs: 456:1261) which targets the pv-lukgene (encoding Panton-Valentine leukocidin) and is included in an 8primer pair panel or kit for use in identification of drug resistantstrains of Staphylococcus aureus.

A representative process flow diagram used for primer selection andvalidation process is outlined in FIG. 1. For each group of organisms,candidate target sequences are identified (200) from which nucleotidealignments are created (210) and analyzed (220). Primers are thendesigned by selecting appropriate priming regions (230) to facilitatethe selection of candidate primer pairs (240). The primer pairs are thensubjected to in silico analysis by electronic PCR (ePCR) (300) whereinbioagent identifying amplicons are obtained from sequence databases suchas GenBank or other sequence collections (310) and checked forspecificity in silico (320). Bioagent identifying amplicons obtainedfrom GenBank sequences (310) can also be analyzed by a probability modelwhich predicts the capability of a given amplicon to identify unknownbioagents such that the base compositions of amplicons with favorableprobability scores are then stored in a base composition database (325).Alternatively, base compositions of the bioagent identifying ampliconsobtained from the primers and GenBank sequences can be directly enteredinto the base composition database (330). Candidate primer pairs (240)are validated by testing their ability to hybridize to target nucleicacid by an in vitro amplification by a method such as PCR analysis (400)of nucleic acid from a collection of organisms (410). Amplificationproducts thus obtained are analyzed by gel electrophoresis or by massspectrometry to confirm the sensitivity, specificity and reproducibilityof the primers used to obtain the amplification products (420).

Many of the important pathogens, including the organisms of greatestconcern as biowarfare agents, have been completely sequenced. Thiseffort has greatly facilitated the design of primers for the detectionof unknown bioagents. The combination of broad-range priming withdivision-wide and drill-down priming has been used very successfully inseveral applications of the technology, including environmentalsurveillance for biowarfare threat agents and clinical sample analysisfor medically important pathogens.

Synthesis of primers is well known and routine in the art. The primersmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed.

In some embodiments primers are employed as compositions for use inmethods for identification of bacterial bioagents as follows: a primerpair composition is contacted with nucleic acid (such as, for example,bacterial DNA or DNA reverse transcribed from the rRNA) of an unknownbacterial bioagent. The nucleic acid is then amplified by a nucleic acidamplification technique, such as PCR for example, to obtain anamplification product that represents a bioagent identifying amplicon.The molecular mass of each strand of the double-stranded amplificationproduct is determined by a molecular mass measurement technique such asmass spectrometry for example, wherein the two strands of thedouble-stranded amplification product are separated during theionization process. In some embodiments, the mass spectrometry iselectrospray Fourier transform ion cyclotron resonance mass spectrometry(ESI-FTICR-MS) or electrospray time of flight mass spectrometry(ESI-TOF-MS). A list of possible base compositions can be generated forthe molecular mass value obtained for each strand and the choice of thecorrect base composition from the list is facilitated by matching thebase composition of one strand with a complementary base composition ofthe other strand. The molecular mass or base composition thus determinedis then compared with a database of molecular masses or basecompositions of analogous bioagent identifying amplicons for known viralbioagents. A match between the molecular mass or base composition of theamplification product and the molecular mass or base composition of ananalogous bioagent identifying amplicon for a known viral bioagentindicates the identity of the unknown bioagent. In some embodiments, theprimer pair used is one of the primer pairs of Table 2. In someembodiments, the method is repeated using one or more different primerpairs to resolve possible ambiguities in the identification process orto improve the confidence level for the identification assignment.

In some embodiments, a bioagent identifying amplicon may be producedusing only a single primer (either the forward or reverse primer of anygiven primer pair), provided an appropriate amplification method ischosen, such as, for example, low stringency single primer PCR(LSSP-PCR). Adaptation of this amplification method in order to producebioagent identifying amplicons can be accomplished by one with ordinaryskill in the art without undue experimentation.

In some embodiments, the oligonucleotide primers are broad range surveyprimers which hybridize to conserved regions of nucleic acid encodingthe hexon gene of all (or between 80% and 100%, between 85% and 100%,between 90% and 100% or between 95% and 100%) known bacteria and producebacterial bioagent identifying amplicons.

In some cases, the molecular mass or base composition of a bacterialbioagent identifying amplicon defined by a broad range survey primerpair does not provide enough resolution to unambiguously identify abacterial bioagent at or below the species level. These cases benefitfrom further analysis of one or more bacterial bioagent identifyingamplicons generated from at least one additional broad range surveyprimer pair or from at least one additional division-wide primer pair.The employment of more than one bioagent identifying amplicon foridentification of a bioagent is herein referred to as triangulationidentification.

In other embodiments, the oligonucleotide primers are division-wideprimers which hybridize to nucleic acid encoding genes of species withina genus of bacteria. In other embodiments, the oligonucleotide primersare drill-down primers which enable the identification of sub-speciescharacteristics. Drill down primers provide the functionality ofproducing bioagent identifying amplicons for drill-down analyses such asstrain typing when contacted with nucleic acid under amplificationconditions. Identification of such sub-species characteristics is oftencritical for determining proper clinical treatment of viral infections.In some embodiments, sub-species characteristics are identified usingonly broad range survey primers and division-wide and drill-down primersare not used.

In some embodiments, the primers used for amplification hybridize to andamplify genomic DNA, and DNA of bacterial plasmids.

In some embodiments, various computer software programs may be used toaid in design of primers for amplification reactions such as PrimerPremier 5 (Premier Biosoft, Palo Alto, Calif.) or OLIGO Primer AnalysisSoftware (Molecular Biology Insights, Cascade, Colo.). These programsallow the user to input desired hybridization conditions such as meltingtemperature of a primer-template duplex for example. In someembodiments, an in silico PCR search algorithm, such as (ePCR) is usedto analyze primer specificity across a plurality of template sequenceswhich can be readily obtained from public sequence databases such asGenBank for example. An existing RNA structure search algorithm (Mackeet al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporatedherein by reference in its entirety) has been modified to include PCRparameters such as hybridization conditions, mismatches, andthermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A.,1998, 95, 1460-1465, which is incorporated herein by reference in itsentirety). This also provides information on primer specificity of theselected primer pairs. In some embodiments, the hybridization conditionsapplied to the algorithm can limit the results of primer specificityobtained from the algorithm. In some embodiments, the meltingtemperature threshold for the primer template duplex is specified to be35° C. or a higher temperature. In some embodiments the number ofacceptable mismatches is specified to be seven mismatches or less. Insome embodiments, the buffer components and concentrations and primerconcentrations may be specified and incorporated into the algorithm, forexample, an appropriate primer concentration is about 250 nM andappropriate buffer components are 50 mM sodium or potassium and 1.5 mMMg²⁺.

One with ordinary skill in the art of design of amplification primerswill recognize that a given primer need not hybridize with 100%complementarity in order to effectively prime the synthesis of acomplementary nucleic acid strand in an amplification reaction.Moreover, a primer may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent. (e.g., for example, a loop structure or a hairpin structure). Theprimers of the present invention may comprise at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99% sequence identity with any of the primers listed in Table 2. Thus,in some embodiments of the present invention, an extent of variation of70% to 100%, or any range therewithin, of the sequence identity ispossible relative to the specific primer sequences disclosed herein.Determination of sequence identity is described in the followingexample: a primer 20 nucleobases in length which is identical to another20 nucleobase primer having two non-identical residues has 18 of 20identical residues (18/20=0.9 or 90% sequence identity). In anotherexample, a primer 15 nucleobases in length having all residues identicalto a 15 nucleobase segment of primer 20 nucleobases in length would have15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for UNIX, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, complementarity of primers with respect to theconserved priming regions of viral nucleic acid is between about 70% andabout 75% 80%. In other embodiments, homology, sequence identity orcomplementarity, is between about 75% and about 80%. In yet otherembodiments, homology, sequence identity or complementarity, is at least85%, at least 90%, at least 92%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or is 100%.

In some embodiments, the primers described herein comprise at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or100% (or any range therewithin) sequence identity with the primersequences specifically disclosed herein.

One with ordinary skill is able to calculate percent sequence identityor percent sequence homology and able to determine, without undueexperimentation, the effects of variation of primer sequence identity onthe function of the primer in its role in priming synthesis of acomplementary strand of nucleic acid for production of an amplificationproduct of a corresponding bioagent identifying amplicon.

In one embodiment, the primers are at least 13 nucleobases in length. Inanother embodiment, the primers are less than 36 nucleobases in length.

In some embodiments of the present invention, the oligonucleotideprimers are 13 to 35 nucleobases in length (13 to 35 linked nucleotideresidues). These embodiments comprise oligonucleotide primers 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34 or 35 nucleobases in length, or any range therewithin. Thepresent invention contemplates using both longer and shorter primers.Furthermore, the primers may also be linked to one or more other desiredmoieties, including, but not limited to, affinity groups, ligands,regions of nucleic acid that are not complementary to the nucleic acidto be amplified, labels, etc. Primers may also form hairpin structures.For example, hairpin primers may be used to amplify short target nucleicacid molecules. The presence of the hairpin may stabilize theamplification complex (see e.g., TAQMAN MicroRNA Assays, AppliedBiosystems, Foster City, Calif.).

In some embodiments, any oligonucleotide primer pair may have one orboth primers with less then 70% sequence homology with a correspondingmember of any of the primer pairs of Table 2 if the primer pair has thecapability of producing an amplification product corresponding to abioagent identifying amplicon. In other embodiments, any oligonucleotideprimer pair may have one or both primers with a length greater than 35nucleobases if the primer pair has the capability of producing anamplification product corresponding to a bioagent identifying amplicon.

In some embodiments, the function of a given primer may be substitutedby a combination of two or more primers segments that hybridize adjacentto each other or that are linked by a nucleic acid loop structure orlinker which allows a polymerase to extend the two or more primers in anamplification reaction.

In some embodiments, the primer pairs used for obtaining bioagentidentifying amplicons are the primer pairs of Table 2. In otherembodiments, other combinations of primer pairs are possible bycombining certain members of the forward primers with certain members ofthe reverse primers. An example can be seen in Table 2 for two primerpair combinations of forward primer 16S_EC_(—)789_(—)810_F (SEQ ID NO:206), with the reverse primers 16S_EC_(—)880_(—)894_R (SEQ ID NO: 796),or 16 S_EC_(—)882_(—)899_R or (SEQ ID NO: 818). Arriving at a favorablealternate combination of primers in a primer depends upon the propertiesof the primer pair, most notably the size of the bioagent identifyingamplicon that would be produced by the primer pair, which preferably isbetween about 45 to about 150 nucleobases in length. Alternatively, abioagent identifying amplicon longer than 150 nucleobases in lengthcould be cleaved into smaller segments by cleavage reagents such aschemical reagents, or restriction enzymes, for example.

In some embodiments, the primers are configured to amplify nucleic acidof a bioagent to produce amplification products that can be measured bymass spectrometry and from whose molecular masses candidate basecompositions can be readily calculated.

In some embodiments, any given primer comprises a modificationcomprising the addition of a non-templated T residue to the 5′ end ofthe primer (i.e., the added T residue does not necessarily hybridize tothe nucleic acid being amplified). The addition of a non-templated Tresidue has an effect of minimizing the addition of non-templatedadenosine residues as a result of the non-specific enzyme activity ofTaq polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), anoccurrence which may lead to ambiguous results arising from molecularmass analysis.

In some embodiments of the present invention, primers may contain one ormore universal bases. Because any variation (due to codon wobble in the3^(rd) position) in the conserved regions among species is likely tooccur in the third position of a DNA (or RNA) triplet, oligonucleotideprimers can be designed such that the nucleotide corresponding to thisposition is a base which can bind to more than one nucleotide, referredto herein as a “universal nucleobase.” For example, under this “wobble”pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C,and uridine (U) binds to U or C. Other examples of universal nucleobasesinclude nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes etal., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degeneratenucleotides dP or dK (Hill et al.), an acyclic nucleoside analogcontaining 5-nitroindazole (Van Aerschot et al., Nucleosides andNucleotides, 1995, 14, 1053-1056) or the purine analog1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al.,Nucl. Acids Res., 1996, 24, 3302-3306).

In some embodiments, to compensate for the somewhat weaker binding bythe wobble base, the oligonucleotide primers are designed such that thefirst and second positions of each triplet are occupied by nucleotideanalogs that bind with greater affinity than the unmodified nucleotide.Examples of these analogs include, but are not limited to,2,6-diaminopurine which binds to thymine, 5-propynyluracil (also knownas propynylated thymine) which binds to adenine and 5-propynylcytosineand phenoxazines, including G-clamp, which binds to G. Propynylatedpyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and5,484,908, each of which is commonly owned and incorporated herein byreference in its entirety. Propynylated primers are described in U.S.Pre-Grant Publication No. 2003-0170682, which is also commonly owned andincorporated herein by reference in its entirety. Phenoxazines aredescribed in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each ofwhich is incorporated herein by reference in its entirety. G-clamps aredescribed in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which isincorporated herein by reference in its entirety.

In some embodiments, primer hybridization is enhanced using primerscontaining 5-propynyl deoxy-cytidine and deoxy-thymidine nucleotides.These modified primers offer increased affinity and base pairingselectivity.

In some embodiments, non-template primer tags are used to increase themelting temperature (T_(m)) of a primer-template duplex in order toimprove amplification efficiency. A non-template tag is at least threeconsecutive A or T nucleotide residues on a primer which are notcomplementary to the template. In any given non-template tag, A can bereplaced by C or G and T can also be replaced by C or G. AlthoughWatson-Crick hybridization is not expected to occur for a non-templatetag relative to the template, the extra hydrogen bond in a G-C pairrelative to an A-T pair confers increased stability of theprimer-template duplex and improves amplification efficiency forsubsequent cycles of amplification when the primers hybridize to strandssynthesized in previous cycles.

In other embodiments, propynylated tags may be used in a manner similarto that of the non-template tag, wherein two or more 5-propynylcytidineor 5-propynyluridine residues replace template matching residues on aprimer. In other embodiments, a primer contains a modifiedinternucleoside linkage such as a phosphorothioate linkage, for example.

In some embodiments, the primers contain mass-modifying tags. Reducingthe total number of possible base compositions of a nucleic acid ofspecific molecular weight provides a means of avoiding a persistentsource of ambiguity in determination of base composition ofamplification products. Addition of mass-modifying tags to certainnucleobases of a given primer will result in simplification of de novodetermination of base composition of a given bioagent identifyingamplicon from its molecular mass.

In some embodiments of the present invention, the mass modifiednucleobase comprises one or more of the following: for example,7-deaza-2′-deoxyadenosine-5-triphosphate,5-iodo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxycytidine-5′-triphosphate,5-iodo-2′-deoxycytidine-5′-triphosphate,5-hydroxy-2′-deoxyuridine-5′-triphosphate,4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate,5-fluoro-2′-deoxyuridine-5′-triphosphate,O6-methyl-2′-deoxyguanosine-5′-triphosphate,N2-methyl-2′-deoxyguanosine-5′-triphosphate,8-oxo-2′-deoxyguanosine-5′-triphosphate orthiothymidine-5′-triphosphate. In some embodiments, the mass-modifiednucleobase comprises ¹⁵N or ¹³C or both ¹⁵N and ¹³C.

In some embodiments, multiplex amplification is performed where multiplebioagent identifying amplicons are amplified with a plurality of primerpairs. The advantages of multiplexing are that fewer reaction containers(for example, wells of a 96- or 384-well plate) are needed for eachmolecular mass measurement, providing time, resource and cost savingsbecause additional bioagent identification data can be obtained within asingle analysis. Multiplex amplification methods are well known to thosewith ordinary skill and can be developed without undue experimentation.However, in some embodiments, one useful and non-obvious step inselecting a plurality candidate bioagent identifying amplicons formultiplex amplification is to ensure that each strand of eachamplification product will be sufficiently different in molecular massthat mass spectral signals will not overlap and lead to ambiguousanalysis results. In some embodiments, a 10 Da difference in mass of twostrands of one or more amplification products is sufficient to avoidoverlap of mass spectral peaks.

In some embodiments, as an alternative to multiplex amplification,single amplification reactions can be pooled before analysis by massspectrometry. In these embodiments, as for multiplex amplificationembodiments, it is useful to select a plurality of candidate bioagentidentifying amplicons to ensure that each strand of each amplificationproduct will be sufficiently different in molecular mass that massspectral signals will not overlap and lead to ambiguous analysisresults.

C Determination of Molecular Mass of Bioagent Identifying Amplicons

In some embodiments, the molecular mass of a given bioagent identifyingamplicon is determined by mass spectrometry. Mass spectrometry hasseveral advantages, not the least of which is high bandwidthcharacterized by the ability to separate (and isolate) many molecularpeaks across a broad range of mass to charge ratio (m/z). Thus massspectrometry is intrinsically a parallel detection scheme without theneed for radioactive or fluorescent labels, since every amplificationproduct is identified by its molecular mass. The current state of theart in mass spectrometry is such that less than femtomole quantities ofmaterial can be readily analyzed to afford information about themolecular contents of the sample. An accurate assessment of themolecular mass of the material can be quickly obtained, irrespective ofwhether the molecular weight of the sample is several hundred, or inexcess of one hundred thousand atomic mass units (amu) or Daltons.

In some embodiments, intact molecular ions are generated fromamplification products using one of a variety of ionization techniquesto convert the sample to gas phase. These ionization methods include,but are not limited to, electrospray ionization (ES), matrix-assistedlaser desorption ionization (MALDI) and fast atom bombardment (FAB).Upon ionization, several peaks are observed from one sample due to theformation of ions with different charges. Averaging the multiplereadings of molecular mass obtained from a single mass spectrum affordsan estimate of molecular mass of the bioagent identifying amplicon.Electrospray ionization mass spectrometry (ESI-MS) is particularlyuseful for very high molecular weight polymers such as proteins andnucleic acids having molecular weights greater than 10 kDa, since ityields a distribution of multiply-charged molecules of the samplewithout causing a significant amount of fragmentation.

The mass detectors used in the methods of the present invention include,but are not limited to, Fourier transform ion cyclotron resonance massspectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole,magnetic sector, Q-TOF, and triple quadrupole.

D. Base Compositions of Bioagent Identifying Amplicons

Although the molecular mass of amplification products obtained usingintelligent primers provides a means for identification of bioagents,conversion of molecular mass data to a base composition signature isuseful for certain analyses. As used herein, “base composition” is theexact number of each nucleobase (A, T, C and G) determined from themolecular mass of a bioagent identifying amplicon. In some embodiments,a base composition provides an index of a specific organism. Basecompositions can be calculated from known sequences of known bioagentidentifying amplicons and can be experimentally determined by measuringthe molecular mass of a given bioagent identifying amplicon, followed bydetermination of all possible base compositions which are consistentwith the measured molecular mass within acceptable experimental error.The following example illustrates determination of base composition froman experimentally obtained molecular mass of a 46-mer amplificationproduct originating at position 1337 of the 16S rRNA of Bacillusanthracis. The forward and reverse strands of the amplification producthave measured molecular masses of 14208 and 14079 Da, respectively. Thepossible base compositions derived from the molecular masses of theforward and reverse strands for the B. anthracis products are listed inTable 1.

TABLE 1 Possible Base Compositions for B. anthracis 46mer AmplificationProduct Calc. Mass Mass Error Base Calc. Mass Mass Error Base ForwardForward Composition of Reverse Reverse Composition of Strand StrandForward Strand Strand Strand Reverse Strand 14208.2935 0.079520 A1 G17C10 T18 14079.2624 0.080600 A0 G14 C13 T19 14208.3160 0.056980 A1 G20C15 T10 14079.2849 0.058060 A0 G17 C18 T11 14208.3386 0.034440 A1 G23C20 T2 14079.3075 0.035520 A0 G20 C23 T3 14208.3074 0.065560 A6 G11 C3T26 14079.2538 0.089180 A5 G5 C1 T35 14208.3300 0.043020 A6 G14 C8 T1814079.2764 0.066640 A5 G8 C6 T27 14208.3525 0.020480 A6 G17 C13 T1014079.2989 0.044100 A5 G11 C11 T19 14208.3751 0.002060 A6 G20 C18 T214079.3214 0.021560 A5 G14 C16 T11 14208.3439 0.029060 A11 G8 C1 T2614079.3440 0.000980 A5 G17 C21 T3 14208.3665 0.006520 A11 G11 C6 T1814079.3129 0.030140 A10 G5 C4 T27 14208.3890 0.016020 A11 G14 C11 T1014079.3354 0.007600 A10 G8 C9 T19 14208.4116 0.038560 A11 G17 C16 T214079.3579 0.014940 A10 G11 C14 T11 14208.4030 0.029980 A16 G8 C4 T1814079.3805 0.037480 A10 G14 C19 T3 14208.4255 0.052520 A16 G11 C9 T1014079.3494 0.006360 A15 G2 C2 T27 14208.4481 0.075060 A16 G14 C14 T214079.3719 0.028900 A15 G5 C7 T19 14208.4395 0.066480 A21 G5 C2 T1814079.3944 0.051440 A15 G8 C12 T11 14208.4620 0.089020 A21 G8 C7 T1014079.4170 0.073980 A15 G11 C17 T3 — — — 14079.4084 0.065400 A20 G2 C5T19 — — — 14079.4309 0.087940 A20 G5 C10 T13

Among the 16 possible base compositions for the forward strand and the18 possible base compositions for the reverse strand that werecalculated, only one pair (shown in bold) are complementary basecompositions, which indicates the true base composition of theamplification product. It should be recognized that this logic isapplicable for determination of base compositions of any bioagentidentifying amplicon, regardless of the class of bioagent from which thecorresponding amplification product was obtained.

In some embodiments, assignment of previously unobserved basecompositions (also known as “true unknown base compositions”) to a givenphylogeny can be accomplished via the use of pattern classifier modelalgorithms. Base compositions, like sequences, vary slightly from strainto strain within species, for example. In some embodiments, the patternclassifier model is the mutational probability model. On otherembodiments, the pattern classifier is the polytope model. Themutational probability model and polytope model are both commonly ownedand described in U.S. patent application Ser. No. 11/073,362 which isincorporated herein by reference in entirety.

In one embodiment, it is possible to manage this diversity by building“base composition probability clouds” around the composition constraintsfor each species. This permits identification of organisms in a fashionsimilar to sequence analysis. A “pseudo four-dimensional plot” can beused to visualize the concept of base composition probability clouds.Optimal primer design requires optimal choice of bioagent identifyingamplicons and maximizes the separation between the base compositionsignatures of individual bioagents. Areas where clouds overlap indicateregions that may result in a misclassification, a problem which isovercome by a triangulation identification process using bioagentidentifying amplicons not affected by overlap of base compositionprobability clouds.

In some embodiments, base composition probability clouds provide themeans for screening potential primer pairs in order to avoid potentialmisclassifications of base compositions. In other embodiments, basecomposition probability clouds provide the means for predicting theidentity of a bioagent whose assigned base composition was notpreviously observed and/or indexed in a bioagent identifying ampliconbase composition database due to evolutionary transitions in its nucleicacid sequence. Thus, in contrast to probe-based techniques, massspectrometry determination of base composition does not require priorknowledge of the composition or sequence in order to make themeasurement.

The present invention provides bioagent classifying information similarto DNA sequencing and phylogenetic analysis at a level sufficient toidentify a given bioagent. Furthermore, the process of determination ofa previously unknown base composition for a given bioagent (for example,in a case where sequence information is unavailable) has downstreamutility by providing additional bioagent indexing information with whichto populate base composition databases. The process of future bioagentidentification is thus greatly improved as more BCS indexes becomeavailable in base composition databases.

E. Triangulation Identification

In some cases, a molecular mass of a single bioagent identifyingamplicon alone does not provide enough resolution to unambiguouslyidentify a given bioagent. The employment of more than one bioagentidentifying amplicon for identification of a bioagent is herein referredto as “triangulation identification.” Triangulation identification ispursued by determining the molecular masses of a plurality of bioagentidentifying amplicons selected within a plurality of housekeeping genes.This process is used to reduce false negative and false positivesignals, and enable reconstruction of the origin of hybrid or otherwiseengineered bioagents. For example, identification of the three parttoxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol.,1999, 87, 270-278) in the absence of the expected signatures from the B.anthracis genome would suggest a genetic engineering event.

In some embodiments, the triangulation identification process can bepursued by characterization of bioagent identifying amplicons in amassively parallel fashion using the polymerase chain reaction (PCR),such as multiplex PCR where multiple primers are employed in the sameamplification reaction mixture, or PCR in multi-well plate formatwherein a different and unique pair of primers is used in multiple wellscontaining otherwise identical reaction mixtures. Such multiplex andmulti-well PCR methods are well known to those with ordinary skill inthe arts of rapid throughput amplification of nucleic acids. In otherrelated embodiments, one PCR reaction per well or container may becarried out, followed by an amplicon pooling step wherein theamplification products of different wells are combined in a single wellor container which is then subjected to molecular mass analysis. Thecombination of pooled amplicons can be chosen such that the expectedranges of molecular masses of individual amplicons are not overlappingand thus will not complicate identification of signals.

F. Codon Base Composition Analysis

In some embodiments of the present invention, one or more nucleotidesubstitutions within a codon of a gene of an infectious organism conferdrug resistance upon an organism which can be determined by codon basecomposition analysis. The organism can be a bacterium, virus, fungus orprotozoan.

In some embodiments, the amplification product containing the codonbeing analyzed is of a length of about 35 to about 200 nucleobases. Theprimers employed in obtaining the amplification product can hybridize toupstream and downstream sequences directly adjacent to the codon, or canhybridize to upstream and downstream sequences one or more sequencepositions away from the codon. The primers may have between about 70% to100% sequence complementarity with the sequence of the gene containingthe codon being analyzed.

In some embodiments, the codon base composition analysis is undertaken

In some embodiments, the codon analysis is undertaken for the purpose ofinvestigating genetic disease in an individual. In other embodiments,the codon analysis is undertaken for the purpose of investigating a drugresistance mutation or any other deleterious mutation in an infectiousorganism such as a bacterium, virus, fungus or protozoan. In someembodiments, the bioagent is a bacterium identified in a biologicalproduct.

In some embodiments, the molecular mass of an amplification productcontaining the codon being analyzed is measured by mass spectrometry.The mass spectrometry can be either electrospray (ESI) mass spectrometryor matrix-assisted laser desorption ionization (MALDI) massspectrometry. Time-of-flight (TOF) is an example of one mode of massspectrometry compatible with the analyses of the present invention.

The methods of the present invention can also be employed to determinethe relative abundance of drug resistant strains of the organism beinganalyzed. Relative abundances can be calculated from amplitudes of massspectral signals with relation to internal calibrants. In someembodiments, known quantities of internal amplification calibrants canbe included in the amplification reactions and abundances of analyteamplification product estimated in relation to the known quantities ofthe calibrants.

In some embodiments, upon identification of one or more drug-resistantstrains of an infectious organism infecting an individual, one or morealternative treatments can be devised to treat the individual.

G. Determination of the Quantity of a Bioagent

In some embodiments, the identity and quantity of an unknown bioagentcan be determined using the process illustrated in FIG. 2. Primers (500)and a known quantity of a calibration polynucleotide (505) are added toa sample containing nucleic acid of an unknown bioagent. The totalnucleic acid in the sample is then subjected to an amplificationreaction (510) to obtain amplification products. The molecular masses ofamplification products are determined (515) from which are obtainedmolecular mass and abundance data. The molecular mass of the bioagentidentifying amplicon (520) provides the means for its identification(525) and the molecular mass of the calibration amplicon obtained fromthe calibration polynucleotide (530) provides the means for itsidentification (535). The abundance data of the bioagent identifyingamplicon is recorded (540) and the abundance data for the calibrationdata is recorded (545), both of which are used in a calculation (550)which determines the quantity of unknown bioagent in the sample.

A sample comprising an unknown bioagent is contacted with a pair ofprimers that provide the means for amplification of nucleic acid fromthe bioagent, and a known quantity of a polynucleotide that comprises acalibration sequence. The nucleic acids of the bioagent and of thecalibration sequence are amplified and the rate of amplification isreasonably assumed to be similar for the nucleic acid of the bioagentand of the calibration sequence. The amplification reaction thenproduces two amplification products: a bioagent identifying amplicon anda calibration amplicon. The bioagent identifying amplicon and thecalibration amplicon should be distinguishable by molecular mass whilebeing amplified at essentially the same rate. Effecting differentialmolecular masses can be accomplished by choosing as a calibrationsequence, a representative bioagent identifying amplicon (from aspecific species of bioagent) and performing, for example, a 2-8nucleobase deletion or insertion within the variable region between thetwo priming sites. The amplified sample containing the bioagentidentifying amplicon and the calibration amplicon is then subjected tomolecular mass analysis by mass spectrometry, for example. The resultingmolecular mass analysis of the nucleic acid of the bioagent and of thecalibration sequence provides molecular mass data and abundance data forthe nucleic acid of the bioagent and of the calibration sequence. Themolecular mass data obtained for the nucleic acid of the bioagentenables identification of the unknown bioagent and the abundance dataenables calculation of the quantity of the bioagent, based on theknowledge of the quantity of calibration polynucleotide contacted withthe sample.

In some embodiments, construction of a standard curve where the amountof calibration polynucleotide spiked into the sample is varied providesadditional resolution and improved confidence for the determination ofthe quantity of bioagent in the sample. The use of standard curves foranalytical determination of molecular quantities is well known to onewith ordinary skill and can be performed without undue experimentation.

In some embodiments, multiplex amplification is performed where multiplebioagent identifying amplicons are amplified with multiple primer pairswhich also amplify the corresponding standard calibration sequences. Inthis or other embodiments, the standard calibration sequences areoptionally included within a single vector which functions as thecalibration polynucleotide. Multiplex amplification methods are wellknown to those with ordinary skill and can be performed without undueexperimentation.

In some embodiments, the calibrant polynucleotide is used as an internalpositive control to confirm that amplification conditions and subsequentanalysis steps are successful in producing a measurable amplicon. Evenin the absence of copies of the genome of a bioagent, the calibrationpolynucleotide should give rise to a calibration amplicon. Failure toproduce a measurable calibration amplicon indicates a failure ofamplification or subsequent analysis step such as amplicon purificationor molecular mass determination. Reaching a conclusion that suchfailures have occurred is in itself, a useful event.

In some embodiments, the calibration sequence is comprised of DNA. Insome embodiments, the calibration sequence is comprised of RNA.

In some embodiments, the calibration sequence is inserted into a vectorthat itself functions as the calibration polynucleotide. In someembodiments, more than one calibration sequence is inserted into thevector that functions as the calibration polynucleotide. Such acalibration polynucleotide is herein termed a “combination calibrationpolynucleotide.” The process of inserting polynucleotides into vectorsis routine to those skilled in the art and can be accomplished withoutundue experimentation. Thus, it should be recognized that thecalibration method should not be limited to the embodiments describedherein. The calibration method can be applied for determination of thequantity of any bioagent identifying amplicon when an appropriatestandard calibrant polynucleotide sequence is designed and used. Theprocess of choosing an appropriate vector for insertion of a calibrantis also a routine operation that can be accomplished by one withordinary skill without undue experimentation.

H. Identification of Bacteria

In other embodiments of the present invention, the primer pairs producebioagent identifying amplicons within stable and highly conservedregions of bacteria. The advantage to characterization of an amplicondefined by priming regions that fall within a highly conserved region isthat there is a low probability that the region will evolve past thepoint of primer recognition, in which case, the primer hybridization ofthe amplification step would fail. Such a primer set is thus useful as abroad range survey-type primer. In another embodiment of the presentinvention, the intelligent primers produce bioagent identifyingamplicons including a region which evolves more quickly than the stableregion described above. The advantage of characterization bioagentidentifying amplicon corresponding to an evolving genomic region is thatit is useful for distinguishing emerging strain variants or the presenceof virulence genes, drug resistance genes, or codon mutations thatinduce drug resistance.

The present invention also has significant advantages as a platform foridentification of diseases caused by emerging bacterial strains such as,for example, drug-resistant strains of Staphylococcus aureus. Thepresent invention eliminates the need for prior knowledge of bioagentsequence to generate hybridization probes. This is possible because themethods are not confounded by naturally occurring evolutionaryvariations occurring in the sequence acting as the template forproduction of the bioagent identifying amplicon. Measurement ofmolecular mass and determination of base composition is accomplished inan unbiased manner without sequence prejudice.

Another embodiment of the present invention also provides a means oftracking the spread of a bacterium, such as a particular drug-resistantstrain when a plurality of samples obtained from different locations areanalyzed by the methods described above in an epidemiological setting.In one embodiment, a plurality of samples from a plurality of differentlocations is analyzed with primer pairs which produce bioagentidentifying amplicons, a subset of which contains a specificdrug-resistant bacterial strain. The corresponding locations of themembers of the drug-resistant strain subset indicate the spread of thespecific drug-resistant strain to the corresponding locations.

I. Kits

The present invention also provides kits for carrying out the methodsdescribed herein. In some embodiments, the kit may comprise a sufficientquantity of one or more primer pairs to perform an amplificationreaction on a target polynucleotide from a bioagent to form a bioagentidentifying amplicon. In some embodiments, the kit may comprise from oneto fifty primer pairs, from one to twenty primer pairs, from one to tenprimer pairs, or from two to five primer pairs. In some embodiments, thekit may comprise one or more primer pairs recited in Table 2.

In some embodiments, the kit comprises one or more broad range surveyprimer(s), division wide primer(s), or drill-down primer(s), or anycombination thereof. If a given problem involves identification of aspecific bioagent, the solution to the problem may require the selectionof a particular combination of primers to provide the solution to theproblem. A kit may be designed so as to comprise particular primer pairsfor identification of a particular bioagent. A drill-down kit may beused, for example, to distinguish different genotypes or strains,drug-resistant, or otherwise. In some embodiments, the primer paircomponents of any of these kits may be additionally combined to compriseadditional combinations of broad range survey primers and division-wideprimers so as to be able to identify a bacterium.

In some embodiments, the kit contains standardized calibrationpolynucleotides for use as internal amplification calibrants. Internalcalibrants are described in commonly owned U.S. patent application Ser.No. 60/545,425 which is incorporated herein by reference in itsentirety.

In some embodiments, the kit comprises a sufficient quantity of reversetranscriptase (if RNA is to be analyzed for example), a DNA polymerase,suitable nucleoside triphosphates (including alternative dNTPs such asinosine or modified dNTPs such as the 5-propynyl pyrimidines or any dNTPcontaining molecular mass-modifying tags such as those described above),a DNA ligase, and/or reaction buffer, or any combination thereof, forthe amplification processes described above. A kit may further includeinstructions pertinent for the particular embodiment of the kit, suchinstructions describing the primer pairs and amplification conditionsfor operation of the method. A kit may also comprise amplificationreaction containers such as microcentrifuge tubes and the like. A kitmay also comprise reagents or other materials for isolating bioagentnucleic acid or bioagent identifying amplicons from amplification,including, for example, detergents, solvents, or ion exchange resinswhich may be linked to magnetic beads. A kit may also comprise a tableof measured or calculated molecular masses and/or base compositions ofbioagents using the primer pairs of the kit.

Some embodiments are kits that contain one or more survey bacterialprimer pairs represented by primer pair compositions wherein each memberof each pair of primers has 70% to 100% sequence identity with thecorresponding member from the group of primer pairs represented by anyof the primer pairs of Table 5. The survey primer pairs may includebroad range primer pairs which hybridize to ribosomal RNA, and may alsoinclude division-wide primer pairs which hybridize to housekeeping genessuch as rplB, tufb, rpoB, rpoC, valS, and infb, for example.

In some embodiments, a kit may contain one or more survey bacterialprimer pairs and one or more triangulation genotyping analysis primerpairs such as the primer pairs of Tables 8, 12, 14, 19, 21, 23, or 24.In some embodiments, the kit may represent a less expansive genotypinganalysis but include triangulation genotyping analysis primer pairs formore than one genus or species of bacteria. For example, a kit forsurveying nosocomial infections at a health care facility may include,for example, one or more broad range survey primer pairs, one or moredivision wide primer pairs, one or more Acinetobacter baumanniitriangulation genotyping analysis primer pairs and one or moreStaphylococcus aureus triangulation genotyping analysis primer pairs.One with ordinary skill will be capable of analyzing in silicoamplification data to determine which primer pairs will be able toprovide optimal identification resolution for the bacterial bioagents ofinterest.

In some embodiments, a kit may be assembled for identification ofstrains of bacteria involved in contamination of food. An example ofsuch a kit embodiment is a kit comprising one or more bacterial surveyprimer pairs of Table 5 with one or more triangulation genotypinganalysis primer pairs of Table 12 which provide strain resolvingcapabilities for identification of specific strains of Campylobacterjejuni.

Some embodiments of the kits are 96-well or 384-well plates with aplurality of wells containing any or all of the following components:dNTPs, buffer salts, Mg²⁺, betaine, and primer pairs. In someembodiments, a polymerase is also included in the plurality of wells ofthe 96-well or 384-well plates.

Some embodiments of the kit contain instructions for PCR and massspectrometry analysis of amplification products obtained using theprimer pairs of the kits.

Some embodiments of the kit include a barcode which uniquely identifiesthe kit and the components contained therein according to productionlots and may also include any other information relative to thecomponents such as concentrations, storage temperatures, etc. Thebarcode may also include analysis information to be read by opticalbarcode readers and sent to a computer controlling amplification,purification and mass spectrometric measurements. In some embodiments,the barcode provides access to a subset of base compositions in a basecomposition database which is in digital communication with basecomposition analysis software such that a base composition measured withprimer pairs from a given kit can be compared with known basecompositions of bioagent identifying amplicons defined by the primerpairs of that kit.

In some embodiments, the kit contains a database of base compositions ofbioagent identifying amplicons defined by the primer pairs of the kit.The database is stored on a convenient computer readable medium such asa compact disk or USB drive, for example.

In some embodiments, the kit includes a computer program stored on acomputer formatted medium (such as a compact disk or portable USB diskdrive, for example) comprising instructions which direct a processor toanalyze data obtained from the use of the primer pairs of the presentinvention. The instructions of the software transform data related toamplification products into a molecular mass or base composition whichis a useful concrete and tangible result used in identification and/orclassification of bioagents. In some embodiments, the kits of thepresent invention contain all of the reagents sufficient to carry outone or more of the methods described herein.

While the present invention has been described with specificity inaccordance with certain of its embodiments, the following examples serveonly to illustrate the invention and are not intended to limit the same.In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner.

EXAMPLES Example 1 Design and Validation of Primers that Define BioagentIdentifying Amplicons for Identification of Bacteria

For design of primers that define bacterial bioagent identifyingamplicons, a series of bacterial genome segment sequences were obtained,aligned and scanned for regions where pairs of PCR primers would amplifyproducts of about 45 to about 150 nucleotides in length and distinguishsubgroups and/or individual strains from each other by their molecularmasses or base compositions. A typical process shown in FIG. 1 isemployed for this type of analysis.

A database of expected base compositions for each primer region wasgenerated using an in silico PCR search algorithm, such as (ePCR). Anexisting RNA structure search algorithm (Macke et al., Nucl. Acids Res.,2001, 29, 4724-4735, which is incorporated herein by reference in itsentirety) has been modified to include PCR parameters such ashybridization conditions, mismatches, and thermodynamic calculations(SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, whichis incorporated herein by reference in its entirety). This also providesinformation on primer specificity of the selected primer pairs.

Table 2 represents a collection of primers (sorted by primer pairnumber) designed to identify bacteria using the methods describedherein. The primer pair number is an in-house database index number.Primer sites were identified on segments of genes, such as, for example,the 16S rRNA gene. The forward or reverse primer name shown in Table 2indicates the gene region of the bacterial genome to which the primerhybridizes relative to a reference sequence. In Table 2, for example,the forward primer name 16 S_EC_(—)1077_(—)1106_F indicates that theforward primer (_F) hybridizes to residues 1077-1106 of the referencesequence represented by a sequence extraction of coordinates4033120.4034661 from GenBank gi number 16127994 (as indicated in Table3). As an additional example: the forward primer nameBONTA_X52066_(—)450_(—)473 indicates that the primer hybridizes toresidues 450-437 of the gene encoding Clostridium botulinum neurotoxintype A (BoNT/A) represented by GenBank Accession No. X52066 (primer pairname codes appearing in Table 2 are defined in Table 3. One withordinary skill knows how to obtain individual gene sequences or portionsthereof from genomic sequences present in GenBank. In Table 2,Tp=5-propynyluracil; Cp=5-propynylcytosine; *=phosphorothioate linkage;I=inosine. T. GenBank Accession Numbers for reference sequences ofbacteria are shown in Table 3 (below). In some cases, the referencesequences are extractions from bacterial genomic sequences orcomplements thereof.

TABLE 2 Primer Pairs for Identification of Bacteria Primer ForwardReverse Pair Forward SEQ Reverse SEQ Number Forward Primer Name SequenceID NO: Reverse Primer Name Sequence ID NO: 1 16S_EC_1077_1106_FGTGAGATGTTG 134 16S_EC_1175_1195_R GACGTCATCCCCA 809 GGTTAAGTCCCCCTTCCTC GTAACGAG 2 16S_EC_1082_1106_F ATGTTGGGTTA 38 16S_EC_1175_1197_RTTGACGTCATCCC 1398 AGTCCCGCAAC CACCTTCCTC GAG 3 16S_EC_1090_1111_FTTAAGTCCCGC 651 16S_EC_1175_1196_R TGACGTCATCCCC 1159 AACGATCGCAAACCTTCCTC 4 16S_EC_1222_1241_F GCTACACACGT 114 16_EC_1303_1323_RCGAGTTGCAGACT 787 GCTACAATG GCGATCCG 5 16S_EC_1332_1353_F AAGTCGGAATC 1016S_EC_1389_1407_R GACGGGCGGTGTG 806 GCTAGTAATCG TACAAG 6 16S_EC_30_54_FTGAACGCTGGT 429 16S_EC_105_126_R TACGCATTACTCA 897 GGCATGCTTAA CCCGTCCGCCAC 7 16S_EC_38_64_F GTGGCATGCCT 136 16S_EC_101_120_R TTACTCACCCGTC 1365AATACATGCAA CGCCGCT GTCG 8 16S_EC_49_68_F TAACACATGCA 15216S_EC_104_120_R TTACTCACCCGTC 1364 AGTCGAACG CGCC 9 16S_EC_683_700_FGTGTAGCGGTG 137 16S_EC_774_795_R GTATCTAATCCTG 839 AAATGCG TTTGCTCCC 1016S_EC_713_732_F AGAACACCGAT 21 16S_EC_789_809_R CGTGGACTACCAG 798GGCGAAGGC GGTATCTA 11 16S_EC_785_806_F GGATTAGAGAC 118 16S_EC_880_897_RGGCCGTACTCCCC 830 CCTGGTAGTCC AGGCG 12 16S_EC_785_810_F GGATTAGATAC 11916S_EC_880_897_2_R GGCGTACTCCCC 830 CCTGGTAGTCA AGGCG CACGC 1316S_EC_789_810_F TAGATACCCTG 206 16S_EC_880_894_R CGTACTCCCCAGG 796GTAGTCCACGC CG 14 16S_EC_960_981_F TTCGATGCAAC 672 16S_EC_1054_1073_RACGAGCTGACGAC 735 GCGAAGAACCT AGCCATG 15 16S_EC_969_985_F ACGCGAAGAAC 1916S_EC_1061_1078_R ACGACCACGAGCTG 734 CTTACC ACGAC 16 23S_EC_1826_1843_FCTGACACCTGC 80 23S_EC_1906_1924_R GACCGTTATAGTT 805 CCGGTGC ACGGCC 1723S_EC_2645_2669_F TCTGTCCCTAG 408 23S_EC_2744_2761_R TGCTTAGATGCTT 1252TACGAGAGGAC TCAGC CGG 18 23S_EC_2645_2669_2_F CTGTCCCTAGT 8323S_EC_2751_2767_R GTTTCATGCTTAG 846 ACGAGAGGACC ATGCTTTCAGC GG 1923S_EC_493_518_F GGGGAGTGAAA 125 23S_EC_551_571_R ACAAAAGGTACGC 717GAGATCCTGAA CGTCACCC ACCG 20 23S_EC_493_518_2_F GGGGAGTGAAA 12523S_EC_551_571_2_R ACAAAAGGCACGC 716 GAGATCCTGAA CATCACCC ACCG 2123S_EC_971_992_F CGAGAGGGAAA 66 23S_EC_1059_1077_R TGGCTGCTTCTAA 1282CAACCCAGACC GCCAAC 22 CAPC_BA_104_131_F GTTATTTAGCA 139CAPC_BA_180_205_R TGAATCTTGAAAC 1150 CTCGTTTTTAA ACCATACGTAAC TCAGCC G23 CAPC_BA_114_133_F ACTCGTTTTTA 20 CAPC_BA_185_205_R TGAATCTTGAAAC 1149ATCAGCCCG ACCATACG 24 CAPC_BA_274_303_F GATTATTGTTA 109CAPC_BA_349_376_R GTAACCCTTGTCT 837 TCCTGTTATGC TTGAATTGTATTT CATTTGAGGC 25 CAPC_BA_276_296_F TTATTGTTATC 663 CAPC_BA_358_377_R GGTAACCCTTGTC834 CTGTTATGCC TTTGAAT 26 CAPC_BA_281_301_F GTTATCCTGTT 138CAPC_BA_361_378_R TGGTAACCCTTGT 1298 ATGCCATTTG CTTTG 27CAPC_BA_315_334_F CCGTGGTATTG 59 CAPC_BA_361_378_R TGGTAACCCTTGT 1298GAGTTATTG TCTTTG 28 CYA_BA_1055_1072_F GAAAGAGTTCG 92 CYA_BA_1112_1130_RTGTTGACCATGCT 1352 GATTGGG TCTTAG 29 CYA_BA_1349_1370_F ACAACGAAGTA 12CYA_BA_1447_1426_R CTTCTACATTTTT 800 CAATACAAGAC AGCCATCAC 30CYA_BA_1353_1379_F CGAAGTACAAT 64 CYA_BA_1448_1467_R TGTTAACGGCTTC 1342ACAAGACAAAA AAGACCC GAAGG 31 CYA_BA_1359_1379_F ACAATACAAGA 13CYA_BA_1447_1461_R CGGCTTCAAGACC 794 CAAAAGAAGG CC 32 CYA_BA_914_937_FCAGGTTTAGTA 53 CYA_BA_999_1026_R ACCACTTTTAATA 728 CCAGAACATGAGGTTTGTAGCTA CAGAC 33 CYA_BA_916_935_F GGTTTAGTACC 131CYA_BA_1003_1025_R CCACTTTTAATAA 768 AGAACATGC GGTTTGTAGC 34INFB_EC_1365_1393_F TGCTCGTGGTG 524 INFB_EC_1439_1467_R TGCTGCTTTCGCA1248 CACAAGTAACG TGGTTAATTGCTT GAT ATTA CAA 35 LEF_BA_1033_1052_FTCAAGAAGAAA 254 LEF_BA_1119_1135_R GAATATCAATTTG 803 AAGAGC TAGC 36LEF_BA_1036_1066_F CAAGAAGAAAA 44 LEF_BA_1119_1149_R AGATAAAGAATCA 745AGAGCTTCTAA CGAATATCAATTT AAAGAATAC GTAGC 37 LEF_BA_756_781_FAGCTTTTGCAT 26 LEF_BA_843_872_R TCTTCCAAGGATA 1135 ATTATATCGAGATTTATTTCTTG CCAC TTCG 38 LEF_BA_758_778_F CTTTTGCATATT 90LEF_BA_843_865_R AGGATAGATTTAT 748 ATATCGAGC TTCTTGTTCG 39LEF_BA_795_813_F TTTACAGCTTT 700 LEF_BA_883_900_R TCTTGACAGCATC 1140ATGCACCG CGTTG 40 LEF_BA_883_899_F CAACGGATGCT 43 LEF_BA_939_958_RCAGATAAAGAATC 762 GGCAAG GCTCCAG 41 PAG_BA_122_142_F CAGAATCAAGT 49PAG_BA_190_209_R CCTGTAGTAGAAG 781 TCCCAGGGG AGGTAAC 42 PAG_BA_123_145_FAGAATCAAGTT 22 PAG_BA_187_210_R CCCTGTAGTAGAA 774 CCCAGGGGTT GAGGTAACCACAC 43 PAG_BA_269_287_F AATCTGCTATT 11 PAG_BA_326_344_R TGATTATCAGCGG1186 TGGTCAGG AAGTAG 44 PAG_BA_655_675_F GAAGGATATAC 93 PAG_BA_755_772_RCCGTGCTCCATTT 778 GGTTGATGTC TTCAG 45 PAG_BA_753_772_F TCCTGAAAAAT 341PAG_BA_849_868_R TCGGATAAGCTGC 1089 GGAGCACGG CACAAGG 46PAG_BA_763_781_F TGGAGCACGG 552 PAG_BA_849_868_R TCGGATAAGCTGC 1089CTTCTGATC CACAAGG 47 RPOC_EC_1018_1045_F CAAACTTATTA 39RPOC_EC_1095_1124_R TCAAGCGCCATTT 959 AGGTAAGCGTG CTTTTGGTAAACC TTGACTACAT 48 RPOC_EC_1018_1045_2_F CAAAACTTATT 39 RPOC_EC_1095_1124_2_RTCAAGCGCCATCT 958 AGGTAAGCGTG CTTTCGGTAATCC TTGACT ACAT 49RPOC_EC_114_140_F TAAGAAGCCGG 158 RPOC_EC_213_232_R GGCGCTTGTACTT 831AAACCATCAAC ACCGCAC TACCG 50 RPOC_EC_2178_2196_F TGATTCTGGTG 478RPOC_EC_2225_2246_R TTGGCCATCAGGC 1414 CCCGTGGT CACGCATAC 51RPOC_EC_2178_2196_2_F TGATTCCGGTG 477 RPOC_EC_2225_2246_2_RTTGGCCATCAGAC 1413 CCCGTGGT CACGCATAC 52 RPOC_EC_2218_2241_F CTGGCAGGTAT81 RPOC_EC_2313_2337_R CGACCGTGGGTT 790 GCGTGGTCTGA GAGATGAAGTAC TG 53RPOC_EC_2218_2241_2_F CTTGCTGGTAT 86 RPOC_EC_2313_2337_2_R CGCACCATGCGTA789 GCGTGGTCTGA GAGATGAAGTAC TG 54 RPOC_EC_808_833_F CGTCGGGTGAT 75RPOC_EC_865_889_R GTTTTTCGTTGCG 847 TAACCGTAACA TACGATGATGTC ACCG 55RPOC_EC_808_833_2_F CGTCGTGTAAT 76 RPOC_EC_865_891_R ACGTTTTTCGTTT 741AACCGTAACA TGAACGATAATGC ACCG T 56 RPOC_EC_993_1019_F CAAAGGTAAGC 41RPOC_EC_1036_1059_R CGAACGGCCTGAG 785 AAGGTCGTTTC TAGTCAACACG CGTCA 57RPOC_EC_993_1019_2_F CAAAGGTAAGC 40 RPOC_EC_1036_1059_2_R CGAACGGCCAGAG784 AAGGACGTTTC TAGTCAACACG CGTCA 58 SSPE_BA_115_137_F CAAGCAAACGC 45SSPE_BA_197_222_R TGCACGTCTGTTT 1201 ACAATCAGAA CAGTTGCAAATTC GC 59TUFB_EC_239_259_F TAGACTGCCCA 204 TUFB_EC_283_303_R GCCGTCCATCTGA 815GGACACGCTG GCAGCACC 60 TUFB_EC_239_259_2_F TTGACTGCCCA 678 TUFB_EC__283_303_2_R GCCGTCCATTTGA 816 GGTCACGCTG GCAGCACC 61 TUFB_EC_976_1000_FAACTACCGTC 4 TUFB_EC_1045_1068_R GTTGTCGCCAGGC 845 CGCAGTTCTACATAACCATTTC TTCC 62 TUFB_EC_976_1000_2_F AACTACCGTCC 5TUFB_EC_1045_1068_2_R GTTGTCACCAGGC 844 TCAGTTCTACT ATTACCATTTC TCC 63TUFB_ECG_985_1012_F CCACAGTTCTA 56 TUFB_EC_1033_1062_R TCCAGGCATTACC1006 CTTCCGTACTA ATTTCTACTCCTT CTGACG CTGG 66 RPLB_EC_650_679_FGACCTACAGTA 98 RPLB_E739_762_R TCCAAGTGCTGGT 999 AGAGGTTCTGT TTACCCCATGGAATTGAACC 67 RPLB_EC_688_710_F CATCCACACGG 54 RFLB_EC_736_757_RGTGCTGGTTTACC 842 TGGTGGTGAAG CCATGGAGT G 68 RPOC_EC_1036_1060_FCGTGTTGACTA 78 RPOC_EC_1097_1126_R ATTCAAGAGCCAT 754 TTCGGGGCGTTCTTCTTTTGGTAAA AG CCAC 69 RPOB_EC_3762_3790_F TCAACAACCTC 248RPOB_EC_3836_3865_R TTTCTTGAAGAGT 1435 TTGGAGGTAAA ATGAGCTGCTCCG GCTCAGTTAAG 70 RPLB_EC_688_710_F CATCCACACGG 54 RPLB_EC_743_771_R TGTTTTGTATCCA1356 TGGTGGTGAAG AGTGCTGGTTTAC G CCC 71 VALS_EC_1105_1124_F CGTGGCGGCGT77 VALS_EC_1195_1218_R CGGTACGAACTGG 795 GGTTATCGA ATGTCGCCGTT 72RPOB_EC_1845_1866_F TATCGCTCAGG 233 RFOB_EC_1909_1929_R GCTGGATTCGCCT825 CGAACTCCAAC TTGCTACG 73 RPLB_EC_669_698_F TGTAATGAACC 623RPLB_BC_735_761_R CCAAGTGCTGGTT 767 CTAATGACCAT TACCCCATGGAGT CCACACGG A74 RPLB_EC_671_700_F TAATGAACCCT 169 RPLB_EC_737_762_R TCCAAGTGCTGGT1000 AATGACCATCC TTACCCCATGGAG 75 SP101_SPET11_1_29_F AACCTTAATTG 2SP101_SPET11_92_116_R CCTACCCAACGTT 779 GAAAGAAACCC CACCAAGGGCAG AAGAAGT76 SP101_SPET11_118_147_F GCTGGTGAAAA 115 SP101_SPET11_213_238_RTGTGGCCGATTTC 1340 TAACCCAGATG ACCACCTGCTCCT TCGTCTTC 77SP101_SPET11_216_243_F AGCAGGTGGTG 24 SP101_SPET11_308_333_RTGCCACTTTGACA 1209 AAATCGGCCAC ACTCCTGTTGCTG ATGATT 78SP101_SPET11_266_295_F CTTGTACTTGT 89 SP101_SFET11_355_380_RGCTGCTTTGATGG 824 GGCTCACACGG CTGAATCCCCTTC CTGTTTGG 79SP101_SPET11_322_344_F GTCAAAGTGGC 132 SF101SFET11_423_441R ATCCCTGCTTCT753 CACGTTTACTG GCTGCC GC 80 SP101_SPET11_358_387_F GGGGATTCAGC 126SP101_SPET11_448_473_R CCAACCTTTTCCA 766 CATCAAAGCAG CAACAGAATCAGCCTATTGAC 81 SP101_SPET11_600_629_F CCTTACTTCGA 62 SP101_SPET11_686_714_RCCCATTTTTTCAC 772 AACTATGAATC GCATGCTGAAAAT TTTTGGAAG ATC 82SP101_SPET11_658_684_F GGGGATTGATA 127 SP101_SPET11_756_784_RGATTGGCGATAAA 813 TCACCATAAG GTGATATTTTCTA AAGAA AAA 83SP101_SPET11_776_801_F TCGCCAATCAA 364 SP101_SPET11_871_896_RGCCCACCAGAAAG 814 AACTAAGGGAA ACTAGCAGGATAA TGGC 84SP101_SPET11_893_921_F GGGCAACAGCA 123 SP101_SPET11_988_1012_RCATGACAGCCAAG 763 GCGGATTGCGA ACCTCACCCACC TTGCGCG 85SP101_SPET11_1154_1179_F CAATACCGCAA 47 SP101_SPET11_1251_1277_RGACCCCAACCTGG 804 CAGCGGTGGCT CCTTTTGTCGTTG TGGG A 86SP101_SPET11_1314_1336_F CGCAAAAAAAT 68 SP101_SPET11_1403_1431_RAAACTATTTTTTT 711 CCAGCTATTAG AGCTATACTCGAA C CAC 87SP101_SPET11_1408_1437_F CGAGTATAGCT 67 SP101_SPET11_1486_1515_RGGATAATTGGTCG 828 AAAAAAATAGT TAACAAGGGATAG TTATGACA TGAG 88SP101_SPET11_1688_1716_F CCTATATTAAT 60 SP101_SPET11_1783_1808_RATATGATTATCAT 752 CGTTTACAGAA TGAACTGCGGCCG ACTGGCT 89SP101_SPET11_1711_1733_F CTGGCTAAAA 82 SP101_SPET11_1808_1835_RGCGTGACAGACCT 821 CTTTGGCAAC TCTTGAATTGTAA GG TCA 90SP101_SPET11_1807_1835_F ATGATTACAAT 33 SP101_SPET11_1901_1927_RTTGGACCTGTAAT 1412 TCAAGAAGGTC CAGCTGAATACTG GTCACGC G 91SP101_SPET11_1967_1991_F TAACGGTTATC 155 SP101_SPET11_2062_2083_RATTGCCCAGAAAT 755 ATGGCCCAGAT CAAATCATC GGG 92 SP101_SPET11_2260_2283_FCAGAGACCGTT 50 SP101_SPET11_2375_2397_R TCTGGGTGACCTG 1131 TTATCCTATCAGTGTTTTAGA GC 93 S9101_SPET11_2375_2399_F TCTAAAACACC 390SP101_SPET11_2470_2497_R AGCTGCTAGATGA 747 AGGTCACCCAG GCTTCTGCCATGG AAGCC 94 SF101_SPET11_2468_2487_F ATGGCCATGGC 35 SP101_SPET11_2543_2570_RCCATAAGGTCACC 770 AGAAGCTCA GTCACCATTCAAA GC 95 SP101_SPET11_2961_2984_FACCATGACAGA 15 SP101_SPET11_3023_3045_R GGAATTTACCAGC 827 AGGCATTTTGAGATAGACACC CA 96 SP101_SPET11_3075_3103_F GATGACTTTTT 108SP101_SPET11_3168_3196_R AATCGACGACCAT 715 AGCTAATGGTC CTTGGAAAGATTTAGGCAGC CTC 97 SP101_SPET11_3386_3403_F AGCGTAAAGGT 25SP101_SPET11_3480_3506_R CCAGCAGTTACTG 769 GAACCTT TCCCCTCATCTTT G 98SP101_SPET11_3511_3535_F GCTTCAGGAAT 116 SP101_SPET11_3605_3629_RGGGTCTACACCTG 832 CAATGATGGAG CACTTGCATAAC CAG 111 RPOB_EC_3775_3803_FCTTGGAGGTAA 87 RFOB_EC_3829_3858_R CGTATAAGCTGCA 797 GTCTCATTTTGCCATAAGCTTGTA GTGGGCA ATGC 112 VALS_EC_1833_1850_F CGACGCGCTGC 65VALS_EC_1920_1943_R GCGTTCCACAGCT 822 GCTTCAC TGTTGCAGAAG 113RPOB_EC_1336_1353_F GACCACCTCGG 97 RPOB_EC_1438_1455_R TTCGCTCTCGGCC1386 CAACCGTA TGGCC 114 TUFB_EC_225_251_F GCACTATGCAC 111TUFB_EC_284_309_R TATAGCACCATCC 930 ACGTAGATTGT ATCTGAGCGGCAC CCTGG 115DNAK_EC_428_449_F CGGCGTACTTC 72 DNAK_EC_503_522_R CGCGGTCGGCTCG 792AACGACAGCCA TTGATGA 116 VALS_EC_1920_1943_F CTTCTGCAACA 85VALS_EC_1948_1970_R TCGCAGTTCATCA 1075 AGCTGTGGAAC GCACGAAGCG GC 117TUFB_EC_757_474_F AAGACGACCTG 6 TUFB_EC_849_867_R GCGCTCCACGTCT 819CACGGGC TCACGC 118 23S_EC_2646_2667_F CTGTTCTTAGT 84 23S_EC_2745_2765_RTTCGTGCTTAGAT 1389 ACGAGAGGACC GCTTTCAG 119 16S_EC_969_985_1P_FACGCGAAGAC 19 16S_EC_1061_1078_2P_R ACGACACGAGCpT 733 CTTACpC pGACGAC120 16S_EC_972_985_2P_F CGAAGAACpCp 63 16S_EC_1064_1075_2P_RACACGAGCpTpGA 727 TTACC C 121 16S_EC_972_985_F CGAAGAACCTT 6316S_EC_1064_1075_R ACACGAGCTGAC 727 ACC 122 TRAN_ILE- CCTGATAAGGG 6123S_SC_40_59_R ACGTCCTTCATCG 740 RRNH_EC_32_50.2_F CCTCTGA 12323S_EC_−7_15_F GTTGTGAGGTT 140 23S_EC_430_450_R CTATCGGTCAGTC 799AAGCGACTAAG AGGAGTAT 124 23S_EC_−7_15_F GTTGTGAGGTT 141 23S_EC_891_910_RTTGCATCGGGTTG 1403 AAGCGACTAAG GTAAGTC 125 23S_EC_430_450_F ATACTCCTGAC30 23S_EC_1424_1442_R AACATAGCCTTCT 712 TGACCGATAG CCGTCC 12623S_EC_891_910_F GACTTACCAAC 100 23S_SC_1908_1931_R TACCTTAGGACCG 893CCGATGCAA TTATAGTTACG 127 23S_EC_1424_1442_F GGACGGAGAAG 11723S_EC_2475_2494_R CCAAACACCGCCG 765 GCTATGTT TCGATAT 12823S_EC_1908_1931_F CGTAACTATAA 73 23S_EC_2833_9852_R GCTTACACACCCG 826CGGTCCTAAGG GCCTATC TA 129 23S_EC_2475_9494_F ATATCGACGGC 31 TRNA_ASP-GCGTGACAGGCAG 820 GGTGTTTGG RRNH_EC_23_41.2_R GTATTC 13116S_EC_−60_−39_F AGTCTCAAGAG 28 16S_EC_508_525_R GCTGCTGGCACGG 823TGAACACGTAA AGTTA 132 16S_EC_326_345_F GACACGGTCCA 95 16S_EC_1041_1058_RCCATGCAGCACCT 771 GACTCCTAC GTCTC 133 16S_EC905_724_F GATCTGGAGGA 10716S_EC_1493_3512_R ACGGTTACCTTGT 739 ATACCGGTG TACGACT 13416S_EC_1268_1287_F GAGAGCAAGCG 101 TRNA_ALA- CCTCCTGCGTGCA 780 GACCTCATARRNH_EC_30_40.2_R AAGC 135 16S_EC_969_985_F ACGCGAAGAAC 1916S_EC_1061_1078.2_R ACAACACGAGCTG 719 CTTACC ACGAC 137 165_EC_969_985_FACGCGAAGAAC 19 16S_EC_1061_1078.2_I14_R ACAACACGAGCTG 721 CCTTACC ICGAC138 165_EC_969_985_F ACGCGAAGAAC 19 16S_EC_1061_107.2_I12_RACAACACGAGCIG 718 CTTACC CGAC 139 16S_EC_969_985_F ACGCGAAGAAC 1916S_EC_1061_1078.2_I11_R ACAACACGAGITG 722 CTTACC ACGAC 14016S_EC_969_985_F ACGCGAAGAAC 19 16S_EC_1061_1078.2_I16_R ACAACACGAGCTG720 CTTACC ACIAC 141 16S_EC_969_985_F ACGCGAAGAAC 1916S_EC_1061_1078.2_2I_R ACAACACGAICTI 723 CTTACC ACGAC 142165_EC_969_985_F ACGCGAAGAAC 19 16S_EC_1061_1078.2_3I_R ACAACACIAICTI724 CTTACC ACGAC 143 16S_EC_99_985_F ACGCGAAGAAC 1916S_EC_1061_1078.2_4I_R ACACCACIAICTI 725 CTTACC ACIAC 14723S_EC_2652_2669_F CTAGTACGAGA 79 23S_EC_2741_2760_R ACTTAGATGCTTT 743GGACCGG CAGCGGT 158 16S_EC_683_700_F GTGTAGCGGTG 137 16S_EC_880_894_RCGTACTCCCCAGG 796 AAATGCG CG 159 16S_EC_1100_1116_F CAACGAGCGCA 4216S_EC_1174_1188_R TCCCCACCTTCCT 1019 ACCCTT CC 215 SSPE_BA_121_137_FAACGCACAATC 3 SSPE_BA_197_216_R TCTGTTTCAGTTG 1132 AGAAGC CAAATTC 220GROL_EC_941_959_F TGGAAGATCTG 544 GROL_EC_1039_1060_R CAATCTGCTGACG 759GGTCAGGC GATCTGAGC 221 INFB_EC_1103_1124_F GTCGTGAAAAC 133INFB_EC_1174_1191_R CATGATGGTCACA 764 GAGCTGGAAGA ACCGG 222HFLB_EC_1082_1102_F TGGCGAACCTG 569 HFLB_EC_1144_1168_R CTTTCGCTTTCTC802 GTGAACGAAGC GAACTCAACCAT 223 INFB_EC_1969_1994_F CGTCAGGGTAA 74INFB_EC_2038_2058_R AACTTCGCCTTCG 713 ATTCCGTGAAG GTCATGTT TTAA 224GROL_EC_219_242_F GGTGAAAGAAG 128 GROL_EC_328_350_R TTCAGGTCCATCG 1377TTGCCCTCTAA GGTTCATGCC AGC 225 VALS_EC_1105_1124_F CGTGGCGGCGT 77VALS_EC_1195_1214_R ACGAACTGGATGT 732 GGTTATCGA CGCCGTT 22616S_EC_556_575_F CGGAATTACTG 70 16S_EC_683_700_R CGCATTTCACCGC 791GGCGTAAAG TACAC 227 RPOC_EC_1256_1277_F ACCCAGTGCTG 16RPOC_EC_1295_1315_R GTTCAAATGCCTG 843 CTGAACCGTGC GATACCCA 22816S_EC_774_795_F GGGAGCAAACA 122 16S_EC_880_894_R CGTACTCCCCAGG 796GGATTAGATAC CG 229 RPOC_EC_1584_1604_F TGGCCCGAAAG 567RPOC_EC_1623_1643_R ACGCGGGCATGCA 737 AAGCTGAGCG GAGATGCC 23016S_EC_1082_1100_F ATGTTGGGTTA 37 16S_EC_1177_1196_R TGACGTCATCCCC 1158AGTCCCGC ACCTTCC 231 16S_EC_1389_1407_F CTTGTACACAC 8816S_EC_1525_1541_R AAGGAGGTGATCC 714 CGCCCGTC AGCC 23216S_EC_1303_1323_F CGGATTGGAGT 71 16S_EC_1389_1407_R GACGGCGGTGTG 808CTGCAACTCG TACAAG 233 23S_EC_23_37_F GGTGGATGCCT 129 23S_EC_115_130_RGGGTTTCCCCATT 833 TGGC CGG 234 23S_EC_187_207_F GGGAACTGAAA 12123S_EC_242_256_R TTCGCTCGCCGCT 1385 CATCTAAGTA AC 235 23S_EC_1602_1620_FTACCCCAAACC 184 23S_EC_1686_1703_R CCTTCTCCCGAAG 782 GACACAGG TTACG 23623S_EC_1685_1703_F CCGTAACTTC 58 23S_EC_1828_1842_R CACCGGGCAGGCG 760GGAGAAGG TC 237 23S_EC_1827_1843_F GACGCCTGCCC 99 23S_EC_1929_1949_RCCGACAAGGAATT 775 GGTGC TCGCTACC 238 23S_EC_2434_2456_F AAGGTACTCCG 923S_EC_9490_2511_R AGCCGACATCGAG 746 GGGATAACAGG GGTGCCAAAC C 23923S_EC_2599_2616_F GACAGTTCGGT 96 23S_SC_2653_2669_R CCGGTCCTCTCGT 777CCCTATC ACTA 240 23S_EC_2653_2669_F TAGTACGAGAG 227 23S_EC_2737_2758_RTTAGATGCTTTCA 1369 GACCGG GCACTTATC 241 23S_ES_−68_−44_F AAACTAGATAA 123S_B_5_21_R GTGCGCCCTTTCT 841 CAGTAGACATC AACTT AC 242 16S_EC_8_27_FAGAGTTTGATC 23 16S_SC_342_358_R ACTGCTGCCTCCC 742 ATGGCTCAG GTAG 24316S_EC_314_332_F CACTGGAACTG 48 16S_EC_556_575_R CTTTACGCCCAGT 801AGACACGG AATTCCG 244 16S_EC_518_536_F CCAGCAGCCGC 57 16S_EC_774_795_RGTATCTAATCCTG 839 GGTAATAC TTTGCTCCC 245 16S_EC_683_700_F GTGTAGCGGTG137 16S_EC_967_985_R GGTAAGGTTCTTC 835 AAATGCG GCGTTG 24616S_EC_937_954_F AAGCGGTGGAG 7 16S_EC_1220_1240_R ATTGTAGCACGTG 757CATGTGG TGTAGCCC 247 16S_EC_1195_1213_F CAAGTCATCAT 4616S_EC_1525_1541_R AAGGAGGTGATCC 714 GGCCCTTA AGCC 248 16S_EC_8_27_FAGAGTTTGATC 23 16S_EC_1525_1541_R AAGGAGGTGATCC 714 ATGGCTCAG AGCC 24923S_EC_1831_1849_F ACCTGCCCAGT 18 23S_EC_1919_1936_R TCGCTACCTTAGG 1080GCTGGAAG ACCGT 250 16S_EC_1387_1407_F GCCTTGTACAC 112 16S_EC_1494_1513_RCACGGCTACCTTG 761 ACCTCCCGTC TTACGAC 251 16S_EC_1390_1411_F TTGTACACACC693 16S_EC_1486_1505_R CCTTGTTACGACT 783 GCCCGTCATAC TCACCCC 25216S_EC_1367_1387_F TACGGTGAATA 191 16S_EC_1485_1506_R ACCTTGTTACGAC 731CGTTCCCGGG TTCACCCCA 253 16S_EC_804_822_F ACCACGCCGTA 1416S_EC_909_929_R CCCCCGTCAATTC 773 AACGATGA CTTTGAGT 25416S_EC_791_812_F GATACCCTGGT 106 16S_EC_886_04_R GCCTTGCGACCGT 817AGTCCACACCG ACTCCC 255 16S_EC_789_810_F TAGATACCCTG 206 16S_EC_882_899_RGCGACCGTACTCC 818 GTAGTCCACGC CCAGG 256 16S_EC_1092_1109_F TAGTCCCGCAA228 16S_EC_1174_1195_R GACGTCATCCCCA 810 CGAGCGC CCTTCCTCC 25723S_EC_2586_2607_F TAGAACGTCGC 203 23S_EC_2658_2677_R AGTCCATCCCGGT 749GAGACAGTTCG CCTCTCG 258 RNASEP_SA_31_49_F GAGGAAAGTCC 103RNASEP_SA_358_379_R ATAAGCCATGTTC 750 ATGCTCAC TGTTCCATC 258RNASEP_SA_31_49_F GAGGAAAGTCC 103 RNASEP_EC_345_362_R ATAAGCCGGGTTC 751ATGCTCAC TGTCG 258 RNASEP_SA_31_49_F GAGGAAAGTCC 103 RNASEP_BS_363_384_RGTAAGCCATGTTT 838 ATGCTCAC TGTTCCATC 258 RNASEP_BS_43_61_F GAGGAAAGTCC104 RNASEP_SA_358_379_R ATAAGCCATGTTC 750 ATGCTCGC TGTTCCATC 258RNASEP_BS_43_61_F GAGGAAAGTCC 104 RNASEP_EC_345_362_R ATAAGCCGGGTTC 751ATGCTCGC TGTCG 258 RNASEP_BS_43_31_F GAGGAAAGTCC 104 RNASEP_ES_363_384_RGTAAGCCATGTTT 838 ATGCTCGC TGTTCCATC 258 RNASEP_EC_61_77_F GAGGAAAGTCC105 RNASEP_SA_358_379_R ATAAGCCATGTTC 750 GGGCTC TGTTCCATC 258RNASEP_EC_61_77_F GAGGAAAGTCC 105 RNASEP_EC_345_362_R ATAAGCCGGGTTC 751GGGCTC TGTCG 258 RNASEP_EC_61_77_F GAGGAAAGTCC 105 RNASEP_BS_363_384_RGTAAGCCATGTTT 838 GGGCTC TGTTCCATC 259 RNASEP_ES_4331_F GAGGAAAGTCC 104RNASEP_ES_363_384_R GTAAGCCATGTTT 838 ATGCTCGC TGTTCCATC 260RNASEP_EC_61_77_F GAGGAAAGTCC 105 RNASEP_EC_345_362_R ATAAGCCGGGTTC 751GGGCTC TGTCG 262 RNASEP_SA_31_49_F GAGGAAAGTCC 103 RNASEP_SA_358_379_RATAAGCCATGTTC 750 ATGCTCAC TGTTCCATC 263 16S_EC_1082_1100_F ATGTTGGGTTA37 16S_EC_1525_1541_R AAGGAGGTGATCC 714 AGTCCCGC AGCC 26416S_EC_556_575_F CGGAATTACTG 70 16S_EC_774_795_R GTATCTAATCCTG 839GGCGTAAAG TTGCTCCC 265 16S_EC_1082_1100_F ATGTTGGGTTA 3716S_EC_1177_1196_10G_R TGACGCATGCCC 1160 AGTCCCGC ACCTTCC 26616S_EC_1082_1100_F ATGTTGGGTTA 37 16S_EC_1177_1196_10G_11G_RTGACGTCATGGCC 1161 AGTCCCGC ACCTTCC 268 YAED_EC_513_532_F_MODGGTGTTAAATA 130 TRNA_ALA- AGACCTCCTGCGT 744 GCCTGGCAGRRNA_EC_30_49_F_MOD GCAAAGC 269 16S_EC_1082_1100_F_MOD ATGTTGGGTTA 3716S_EC_1177_1196_R_MOD TGACGCATCCCC 1158 AGTCCCGC ACCTTCC 27023S_EC_2586_2607_F_MOD TAGAACGTCGC 203 23S_EC_2658_2677_R_MODAGTCCATCCCGGT 749 GAGACAGTTCG CCTCTCG 272 16S_EC_969_985_F ACGCGAAGAAC19 16S_EC_1389_1407_R GACGGGCGGTGTG 807 CTTACC TACAAG 27316S_EC_683_700_F GTGTAGCGGTG 137 16S_EC_1303_1323_R CGAGTTGCAGACT 788AAATGCG GCGATCCG 274 169_EC_49_68_F TAACACATGCA 152 16S_EC_880_894_RCGTACTCCCCAGG 796 AGTCGAACG CG 275 16S_EC_49_68_F TAACACATGCA 15216S_EC_1061_1078_R ACGACACGAGCTG 734 AGTCGAACG ACGAC 277CYA_BA_1349_1370_F ACAACGAAGTA 12 CYA_BA_1426_1447_R CTTCTACATTTTT 800CAATACAAGAC AGCCATCAC 278 16S_EC_1090_1111_2_F TTAAGTCCCGC 65016S_EC_1175_1196_R TGACGTCATCCCC 1159 AACGAGCGCAA ACCTTCCTC 27916S_EC_405_432_F TGAGTGATGAA 464 16S_EC_507_527_R CGGCTGCTGGCAC 793GGCCTTAGGGT GAAGTTAG TGTAAA 280 GROL_EC_496_518_F ATGGACAAGGT 34GROL_EC_577_596_R TAGCCGCGGTCGA 914 TGGCAAGGAAG ATTGCAT G 281GROL_EC_511_536_F AAGGAAGGCGT 8 GROL_EC_571_593_R CCGCGGTCGAATT 776GATCACCGTTG GCATGCCTTC AAGA 288 RPOB_EC_3802_3821_F CAGCGTTTCGG 51RPOB_EC_3862_3885_R CGACTTGACGGTT 786 CGAAATGGA AACATTTCCTG 289RPOB_EC_3799_3821_F GGGCAGCGTTT 124 RPOB_EC_3862_3888_R GTCCGACTTGACG840 CGGCGAAATG GTCAACATTTCCT GA G 290 RPOC_EC_2146_2174_F CAGGAGTCGTT 52RPOC_EC_2227_2245_R ACGCCATCAGGCC 736 CAACTCGATCT ACGCAT ACATGAT 291ASPS_EC_405_422_F GCACAACCTGC 110 ASPS_EC_521_538_R ACGGCACGAGGTA 738GGCTGCG GTCGC 292 RPOC_EC_1374_1393_F CGCCGACTTCG 69 RPOC_EC_1437_1455_RGAGCATCAGCGTG 811 ACGGTGACC CGTGCT 293 TUFB_EC_957_979_F CCACACGCCGT 55TUFB_EC_1034_1058_R GGCATCACCATTT 829 TCTTCAACAAC CCTTGTCCTTCG T 29416S_EC_7_33_F GAGAGTTTGAT 102 16S_EC_101_122_R TGTTACTCACCCG 1345CCTGGCTCAGA TCTGCCACT ACGAA 295 VALS_EC_610_649_F ACCGAGCAAGG 17VALS_EC_705_727_R TATAACGCACATC 929 AGACCAGC GTCAGGGTGA 34416S_EC_971_990_F GCGAAGAACCT 113 16S_EC_1043_1062_R ACAACCATGCACC 726TACCAGGTC ACCTGTC 346 16S_EC_713_732_TMOD_F TAGAACACCGA 20216S_EC_789_809_TMOD_R TCGTGGACTACCA 1110 TGGCGAAGGC GGGTATCTA 34716S_EC_785_806_TMOD_F TGGATTAGAGA 560 16S_EC_880_897_TMOD_RTGGCCGTACTCCC 1278 CCCTGGTAGTC CAGGCG C 348 16S_EC_960_981_TMOD_FTTTCGATGCAA 706 16S_EC_1054_1073_TMOD_R TACGAGCTGACGA 895 CGCGAAGAACCCAGCCATG T 349 23S_EC_1826_1843_TMOD_F TCTGACACCTG 40123S_EC_1906_1924_TMOD_R TGACCGTTATAGT 1156 CCCGGTGC TACGGCC 350CAPC_BA_274_303_TMOD_F TGATTATTGTT 476 CAPC_BA_349_376_TMOD_RTGTAACCCTTGTC 1314 ATCCTGTTATG TTTGAATTGTAT CCATTTGAG TGC 351CYA_BA_1353_1379_TMOD_F TCGAAGTACAA 355 CYA_BA_1448_1467_TMOD_RTTGTTAACGGCTT 1423 TACAAGACAAA CAAGACCC AGAAGG 352INFB_EC_1365_1393_TMOD_F TTGCTCGTGGT 687 INFB_EC_1439_1467_TMOD_RTTGCTGCTTTCGC 1411 GCACAAGTAAC ATGGTTAATTGCT GGATATTA TCAA 353LEF_BA_756_781_TMOD_F TAGCTTTTGCA 220 LEF_BA_843_872_TMOD_RTTCTTCCAAGGAT 1394 TATTATATCGA AGATTTATTTCTT GCCAC GTTCG 354RPOC_EC_2218_2241_TMOD_F TCTGGCAGGTA 405 RPOC_EC_2313_2337_TMOD_RTCGCACCGTGGGT 1072 TGCGTGGTCTG TGAGATGAAGTAC ATG 355SSPE_BA_115_137_TMOD_F TCAAGCAAACG 255 SSPE_BA_197_222_TMOD_RTTGCACGTCTGTT 1402 CACAATCAGAA TCAGTTGCAAATT GC C 356RPLB_EC_650_679_TMOD_F TGACCTACAGT 449 RPLB_EC_739_762_TMOD_RTTCCAAGTGCTGG 1380 AAGAGGTTCTG TTTACCCCATGG TAATGAACC 357RPLB_EC_688_710_TMOD_F TCATCCACACG 296 RPLB_EC_736_757_TMOD_RTGTGCTGGTTTAC 1337 GTGGTGGTGAA CCCATGGAGT GG 358VALS_EC_1105_1124_TMOD_F TCGTGGCGGCG 385 VALS_EC_1195_1218_TMOD_RTCGGTACGAACTG 1093 TGGTTATCGA GATGTCGCCGTT 359 RPOB_EC_1845_1866_TMOD_FTTATCGCTCAG 659 RPOB_EC_1909_1929_TMOD_R TGCTGGATTCGCC 1250 GCGAACTCCAATTTGCTACG C 360 23SEC_2646_2667_TMOD_F TCTGTTCTTAG 40923S_EC_2745_2765_TMOD_R TTTCGTGCTTAGA 1434 TACGAGAGGAC TGCTTTCAG C 36116S_EC_1090_1111_2_TMOD_F TTTAAGTCCCG 697 16S_EC_1175_1196_TMOD_RTTGACGTCATCCC 1398 CAACGAGCGCA CACCTTCCTC A 362 RPOB_EC_3799_3821_TMOD_FTGGGCAGCGTT 581 RPOB_EC_3862_3888_TMOD_R TGTCCGACTTGAC 1325 TCGGCGAAATGGGTCAACATTTCC GA TG 363 RPOC_EC_2146_2174_TMOD_F TCAGGAGTCGT 284EPOC_EC_2227_2245_TMOD_R TACGCCATCAGGC 898 TCAACTCGATC CACGCAT TACATGAT364 RPOC_EC_1374_1393_TMOD_F TCGCCGACTTC 367 RPOC_EC_1437_1455_TMOD_RTGAGCATCAGCGT 1166 GACGGTGACC GCGTGCT 367 TUFB_ECG_957_979_TMOD_FTCCACACGCCG 308 TUFB_EC_1034_1058_TMOD_R TGGCATCACCATT 1276 TTCTTCAACAATCCTTGTCCTTCG CT 423 SP101_SPET11_893_921_TMOD_F TGGGCAACAGC 580SP101_SPET11_988_1012_TMOD_R TCATGACAGCCAA 990 AGCGGATTGCG GACCTCACCCACCATTGCGCG 424 SP101_SPET11_1154_1179_(—) TCAATACCGCA 258SP101_SPET11_1251_1277_TMOD_R TGACCCCAACCTG 1155 TMOD_F ACAGCGGTGGCGCCTTTTGTCGTT TTGGG GA 425 SP101_SPET11_118_147_TMOD_F TGCTGGTGAAA 528SP101_SPET11_213_238_TMOD_R TTGTGGCCGATTT 1422 ATAACCCAGAT CACCACCTGCTCCGTCGTCTTC T 426 SP101_SPET11_1314_1336_TMOD_F TCGCAAAAAAA 363SP101_SPET11_1403_1431_TMOD_R TAAACTATTTTTT 849 TCCAGCTATTATAGCTATACTCGA GC ACAC 427 SP101_SPET11_1408_1437_(—) TCGAGTATAGC 359SP101_SPET11_1486_1515_TMOD_R TGGATAATTGGTC 1268 TMOD_F TTAAAAAAATAGTAACAAGGGATA GTTTATGACA GTGAG 428 SP101_SPET11_1688_1716_TMOD_FTCCTATATTAA 334 SP101_SPET11_1783_1808_TMOD_R TATATGATTATCA 932TCGTTTACAGA TTGAACTGCGGCC AACTGGCT G 429 SP101_SPET11_1711_1733_(—)TCTGGCTAAAA 406 SP101_SPET11_1808_1835_TMOD_R TGCGTGACGACCT 1239 TMOD_FCTTTGGCAACG TCTTGAATTGTAA GT TCA 430 SP101_SPET11_1807_1835_TMOD_FTATGATTACAA 235 SP101_SPET11_1901_1927_TMOD_R TTTGGACCTGTAA 1439TTCAAGAAGGT TCAGCTGAATACT CGTCACGC GG 431 SP101_SPET11_1967_1991_(—)TTAACGGTTAT 649 SP101_SPET11_2082_2083_TMOD_R TATTGCCCAGAAA 940 TMOD_FCATGGCCCAGA TCAAATCATC TGGG 432 SP101_SPET11_216_243_TMOD_F TAGCAGGTGGT210 SP101_SPET11_308_333_TMOD_R TTGCCACTTTGAC 1404 GAAATCGGCCAAACTCCTGTTGCT CATGATT G 433 SP101_SPET11_2260_2283_(—) TCAGAGACCGT 272SP101_SPET11_2375_2397_TMOD_R TTCTGGGTGACCT 1393 TMOD_F TTTATCCTATCGGTGTTTTAGA AGC 434 SP101_SPET11_2375_2399_(—) TTCTAAAACAC 675SP101_SPET11_2470_2497_TMOD_R TAGCTGCTAGATG 918 TMOD_F CAGGTCACCCAAGCTTCTGCCATG GAAG GCC 435 SP101_SPET11_2468_2487_(—) TATGGCCATGG 238SP101_SPET11_2543_2579_TMOD_R TCCATAAGGTCAC 1007 TMOD_F CAGAAGCTCACGTCACCATTCAA AGC 436 SP101_SFET11_266_295_TMOD_F TCTTGTACTTG 417SP101_SPET11_355_380_TMOD_R TGCTGCTTTGATG 1249 TGGCTCACACG GCTGAATCCCCTTGCTGTTTGG C 437 SP101_SPET11_2961_2984_(—) TACCATGACAG 183SP101_SPET11_3023_3045_TMOD_R TGGAATTTACCAG 1264 TMOD_F AAGGCATTTTGCGATAGACACC ACA 438 SP101_SPET11_3075_3103_(—) TGATGACTTTT 473SP101_SPET11_3168_3196_TMOD_R TAATCGACGACCA 875 TMOD_F TAGCTAATGGTTCTTGGAAAGATT CAGGCAGC TCTC 439 SP101_SPET11_322_344_TMOD_F TGTCAAAGTGG631 SP101_SPET11_423_441_TMOD_R TATCCCCTGCTTC 934 CACGTTTACTG TGCTGCC GC440 SP101_SPET11_3386_3403_(—) TAGCGTAAAGG 215SP101_SPET11_3480_3506_TMOD_R TCCAGCAGTTACT 1005 TMOD_F TGAACCTTGTCCCCTCATCTT TG 441 SP101_SPET11_3511_3535_(—) TGCTTCAGGAA 531SP101_SPET11_3605_3629_TMOD_R TGGGTCTACACCT 1294 TMOD_F TCAATGATGGAGCACTTGCATAAC GCAG 442 SP101_SPET11_358_387_TMOD_F TGGGGATTCAG 588SP101_SPET11_448_473_TMOD_R TCCAACCTTTTCC 998 CCATCAAAGCA ACAACAGAATCAGGCTATTGAC C 443 SP101_SPET11_600_628_TMOD_F TCCTTACTTCG 348SP101_SPET11_886_714_TMOD_R TCCCATTTTTTCA 1018 AACTATGAATC CGCATGCTGAAAATTTTGGAAG TATC 444 SP101_SPET11_658_684_TMOD_F TGGGGATTGAT 589SP101_SPET11_756_784_TMOD_R TGATTGGCGATAA 1189 ATCACCGATAA AGTGATATTTTCTGAAGAA AAAA 445 SP101_SPET11_776_801_TMOD_F TTCGCCAATCA 673SP101_SPET11_871_896_TMOD_R TGCCCACCAGAAA 1217 AAACTAAGGGA GACTAGCAGGATAATGGC A 446 SP101_SPET11_1_29_TMOD_F TAACCTTAATT 154SP101_SPET11_92_116_TMOD_R TCCTACCCAACGT 1044 GGAAAGAAACC TCACCAAGGGCAGCAAGAAGT 447 SP101_SPET11_364_385_F TCAGCCATCAA 276SP101_SPET11_448_471_R TACCTTTTCCACA 894 AGCAGCTATTG ACAGAATCAGC 448SP101_SPET11_3085_3104_F TAGCTAATGGT 216 SP101_SPET11_3170_3194_RTCGACGACCATCT 1066 CAGGCAGCC TGGAAAGATTTC 449 RPLB_EC_990_710_FTCCACACGGTG 309 RPLB_EC_737_758_R TGTGCTGGTTTAC 1336 GTGGTGAAGGCCCATGGAG 481 BONTA_X52066_538_552_F TATGGCTCTAC 239BONTA_X52066_647_660_R TGTTACTGCTGGA 1346 TCAA T 482BONTA_X5206_538_552P_F TA*TpGGC*Tp 143 BONTA_X52066_647_660P_RTG*Tp*TpA*Cp* 1146 *Cp*TpA*Cp* TpG*Cp*TpGGAT Tp*CpAA 483BONTA_X5206_701_720_F GAATAGCAATT 94 BONTA_X5206_759_775_R TTACTTCTAACCC1367 AATCCAAAT ACTC 484 BONTA_X52066_701_720P_F GAA*TpAG*Cp 91BONTA_X52066_759_775P_R TTA*Cp*Tp*Tp* 1359 AA*Tp*TpAA* Cp*TpAA*Cp*CpTp*Cp*CpAAAT *CpA*Cp*TpC 485 BONTA_X5206_450_473_F TCTAGTAATAA 393BONTA_X5206_517_539_R TAACCATTTCGCG 859 TAGGACCCTCA TAAGATTCAA GC 486BONTA_X52066_450_473P_F T*Cp*TpAGTA 142 BONTA_X5206_517_539P_RTAACCA*Tp*Tp* 857 ATAATAGGA*C Tp*CpGCGTAAGA pCp*Cp*Tp* *Tp*Tp*CpAA C 487BONTA_X52066_591_620_F TGAGTCACTTG 463 BONTA_X52066_644_671_RTCATGTGCTAATG 992 AAGTTGATACA TTACTGCTGGATC AATCCTCT TG 608SSPE_BA_156_168P_F TGGTpGCpTpA 616 SSPE_BA_243_255P_R TGCpAGCpTGATp 1241GCpATT TpGT 609 SSPE_BA_75_89P_F TACpAGAGTpT 192 SSPE_BA_163_177P_RTGTGCTpTpTpGA 1338 pTpGCpGAC ATpGCpT 610 SSPE_BA_150_168P_F TGCTTCTGGTp533 SSPE_BA_243_264P_R TGATTGTTTTGCp 1191 GCpTpAGCpAT AGCpTGATpTpGT T611 SSPE_BA_72_89P_F TGGTACpAGAG 602 SSPE_BA_163_182P_R TCATTTGTGCTpT995 TpTpTpGCpGA pTpGAATpGCpT C 612 SSPE_BA_114_137P_F TCAAGCAAACG 255SSPE_BA_196_222P_R TTGCACGTCpCpG 1401 CACAATpCpAG TTTCAGTTGCAAA AAGC TTC699 SSPE_BA_123_153_F TGCACAATCAG 488 SSPE_BA_202_231_R TTTCACAGCATGC1431 AAGCTAAGAAA ACGTCTGTTTCAG GCGCAAGCT TTGC 700 SSPE_BA_156_168_FTGGTGCTAGCA 612 SSPE_BA_243_255_R TGCAGCTGATTGT 1202 TT 701SSPE_BA_75_89_F TACAGAGTTTG 179 SSPE_BA_163_177_R TGTGCTTTGAATG 1338CGAC CT 702 SSPE_BA_150_168_F TGCTTTCTGGT 533 SSPE_BA_243_264_RTGATTGTTTTGCA 1190 GCTAGCATT GCTGATTGT 703 SSPE_BA_72_89_F TGGTACAGAGT600 SSPE_BA_163_182_R TCATTTGTGCTTT 995 TTGCGAC GAATGCT 704SSPE_BA_146_168_F TGCAAGCTTCT 484 SSPE_BA_242_267_R TTGTGATTGTTTT 1421GGTGCTAGCAT GCAGCTGATTGTG T 705 SSPE_BA_63_89_F TGCTAGTTATG 518SSPE_BA_163_191_R TCATAACTAGCAT 986 GTACAGAGTTT TTGTGCTTTGAAT GCGAC GCT706 SSPE_BA_114_137_F TCAAGCAAACG 255 SSPE_BA_196_222_R TTGCACGTCTGTT1402 CACAATCAGAA TCAGTTGCAAAT GC C 770 PLA_AF053945_7377_7402_FTGACATCCGGC 442 PLA_AF053945_7434_7462_R TCTAAATTCCGCA 1313 TCACGTTATTAAAGACTTTGGCAT TGGT TA 771 PLA_AF053945_7382_7404_F TCCGGCTCACG 327PLA_AF053945_7482_7502_R TGGTCTGAGTACC 1304 TTATTATGGTA TCCTTTGC C 772PLA_AF053945_7481_7503_F TGCAAAGGAGG 481 PLA_AF053945_7539_7562_RTATTGGAAATACC 943 TACTCAGACCA GGCAGCATCTC T 773 PLA_AF053945_7186_7211_FTTATACCGGAA 657 PLA_AF053945_7257_7280_R TAATGCGATACTG 879 ACTTCCCGAAAGCCTGCAAGTC GGAG 774 CAF1_AF053947_33407_33430_F TCAGTTCCGTT 292CAF1_AF053947_33494_33514_R TGCGGGCTGGTTC 1235 ATCGCCATTGC AACAAGAG AT775 CAF1_AF053947_33515_33541_F TCACTCTTACA 270CAF1_AF053947_33595_33621_R TCCTGTTTTATAG 1053 TATAAGGAAGG CCGCCAAGAGTAACGCTC G 776 CAF1_AF053947_33435_33457_F TGGAACTATTG 542CAF1_AF053947_33499_33517_R TGATGCGGGCTGG 1183 CAACTGCTAAT TTCAAC G 777CAF1_AF053947_33687_33716_F TCAGGATGGAA 286 CAF1_AF053947_33755_33782_RTCAAGGTTCTCAC 962 ATAACCACCAA CGTTTACCTTAGG TTCACTAC AG 778INV_U22457_515_539_F TGGCTCCTTGG 573 INV_U22457_571_598_R TGTTAAGTGTGTT1343 TATGACTCTGC GCGGCTGTCTTTA TTC TT 779 INV_U22457_699_724_FTGCTGAGGCCT 525 INV_U22457_753_776_R TCACGCGACGAGT 976 GGACCGATTATGCCATCCATTG TTAC 780 INV_U22457_834_858_F TTATTTACCTG 664INV_U22457_942_966_R TGACCCAAAGCTG 1154 CACTCCCACAA AAAGCTTTACTG CTG 781INV_U22457_1558_1581_F TGGTAACAGAG 597 INV_U22457_1619_1643_RTTGCGTTGCAGAT 1408 CCTTATAGGCG TATCTTTACCAA CA 782LL_NC003143_2366996_(—) TGTACCCGCTAA 627 LL_NC003143_2367073_2367097_RTCTCATCCCGATA 1123 2367019_F AGCACTACCAT TTACCGCCATGA CC 783LL_NC003143_2367172_(—) TGGACGGCATC 550 LL_NC003143_2367249_23672TGGCAACAGCTCA 1272 2367194_F ACGATTCTCTA ACACCTTTGG C 874RPLB_EC_649_679_F TGICCIACIGT 620 RPLB_EC_739_762_TMOD_R TTCCAAGTGCTGG1380 IIGIGGTTCTG TTTACCCCATGG TAATGAACC 875 RPLB_EC_642_679P_FTpCpCpTpTpG 646 RPLB_EC_739_762_TMOD_R TTCCAAGTGCTGG 1380 ITpGICCIACITTTACCCCATGG GTIIGIGGTTC TGTAATGAACC 876 MECIA_Y14051_3315_3341_FTTACACATATC 653 MECIA_Y14051_3367_3393_R TGTGATATGGAGG 1333 GTGAGCAATGATGTAGAAGGTGTT ACTGA A 877 MECA_Y14051_3774_3802_F TAAAACAAACT 144MECA_Y14051_3828_3854_R TCCCAATCTAACT 1015 ACGGTAACATT TCCACATACCATCGATCGCA T 878 MECA_Y14051_3645_3670_F TGAAGTAGAAA 434MECA_Y14051_3690_3719_R TGATCCTGAATGT 1181 TGACTGAACGT TTATATCTTTAACCCGA GCCT 879 MECA_Y14051_4507_4530_F TCAGGTACTGC 288MECA_Y14051_4555_4581_R TGGATAGACGTCA 1269 TATCCACCCTC TATGAAGGTGTGC AAT 880 MECA_Y14051_4510_4530_F TGTACTGCTAT 626 MECA_Y14051_4586_4610_RTATTCTTCGTTAC 939 CCACCCTCAA TCATGCCATACA 881 MECA_Y14051_4669_4698_FTCACCAGGTTC 262 MECA_Y14051_4765_4793_R TAACCACCCCAAG 858 AACTCAAAAAAATTTATCTTTTG ATATTAACA CCA 882 MECA_Y14051_4520_4530P_F TCpCpACpCpC 389MECA_Y14051_4590_4600P_R TpACpTpCpATpG 1357 pTpCpAA CpCpA 883MECA_Y14051_4520_4530P_F TCpCpACpCpC 389 MECA_Y14051_4600_4610P_RTpATpTpCpTpTp 1358 pTpCpAA CpGTpT 902 TRPE_AY094355_1467_1491_FATGTCGATTGC 36 TRPE_AY094355_1569_1592_R TGCGCGAGCTTT 1231 AATCCGTACTTTATTTGGGTTTC GTG 903 TRPE_AY094355_1445_1471_F TGGATGGCATG 557TRPE_AY094355_1551_1580_R TATTTGGGTTTCA 944 GTGAAATGGAT TTCCACTCAGATTATGTC CT 904 TRPE_AY094355_1278_1303_F TCAAATGTACA 247TRPE_AY094355_1392_1418_R TCCTCTTTTCACA 1048 AGGTGAAGTGC GGCTCTACTTCATGTGA C 905 TRPE_AY094355_1064_1086_F TCGACCTTTGG 357TRPE_AY094355_1171_1196_R TACATCGTTTCGC 885 CAGGAACTAGA CCAAGATCAATCA C906 TRPE_AY094355_666_688_F GTGCATGCGGA 135 TRPE_AY094355_769_791_RTTCAAAATGCGGA 1372 TACAGAGCAGA GGCGTATGTG 907 TRPE_AY094355_757_776_FTGCAAGCGCGA 483 TRPE_AY094355_864_883_R TGCCCAGGTACAA 1218 CCACATACGCCTGCAT 908 RECA_AF251469_43_68_F TGGTACATGTG 601RECA_AF251469_140_163_R TTCAAGTGCTTGC 1375 CCTTCATTGAT TCACCATTGTC GCTG909 RECA_AF251469_169_190_F TGACATGCTTG 446 RECA_AF251469_277_300_RTGGCTCATAAGAC 1280 TCCGTTCAGGC GCGCTTGTAGA 910 PARC_X95819_87_110_FTGGTGACTCGG 609 PARC_X95819_201_222_R TTCGGTATAACGC 1387 CATGTTATGAAATCGCAGCA GC 911 PARC_X95819_87_110_F TGGTGACTCGG 609PARC_X95819_192_219_R GGTATAACGCATG 836 CATGTTATGAA GCAGCAAAAGATT GC TA912 PARC_X95819_123_147_F GGCTCAGCCAT 120 PARC_X95819_232_260_RTCGCTCAGCAATA 1081 TTAGTTACCGC ATTCACTATAAGC TAT CGA 913PARC_X95819_43_63_F TCAGCGCGTAC 277 PARC_X95819_143_170_R TTCCCCTGACCTT1383 AGTGGGTGAT CGATTAAAGGATA GC 914 OMPA_AY485227_272_301_F TTACTCCATTA655 OMPA_AY485227_364_388_R GAGCTGCGCCAAC 812 TTGCTTGGTTA GAATAAATCGTCCACTTTCC 915 OMPA_AY485227_379_401_F TGCGCAGCTCT 509OMPA_AY485227_492_519_R TGCCGTAACATAG 1223 TGGTATCGAGT AAGTTACCGTTGA T T916 OMPA_AY485227_311_335_F TACACAACAAT 178 OMPA_AY485227_424_453_RTACGTCGCCTTA 901 GGCGGTAAAGA ACTTGGTTATATT TGG CAGC 917OMPA_AY485227_415_441_F TGCCTCGAAGC 506 OMPA_AY485227_514_546_RTCGGGCGTAGTTT 1092 TGAATATAACC TTAGTAATTAAAT AAGTT CAGAAGT 918OMPA_AY485227_494_520_F TCAACGGTAAC 252 OMPA_AY485227_569_596_RTCGTCGTATTTAT 1108 TTCTATGTTAC AGTGACCAGCACC TTCTG TA 919OMPA_AY485227_551_577_F TCAAGCCGTAC 257 OMPA_AY485227_658_680_RTTTAAGCGCCAGA 1425 GTATTATTAGG AAGCACCAAC TGCTG 920OMPA_AY485227_555_581_F TCCGTACGTAT 328 OMPA_AY485227_635_662_RTCAACACCAGCGT 954 TATTAGGTGCT TACCTAAAGTACC GGTCA TT 921OMPA_AY485227_556_583_F TCGTACGTATT 379 OMPA_Y485227_659_683_RTCGTTTAAGCGCC 1114 ATTAGGTGCTG AGAAAGCACCAA GTCACT 922OMPA_AY485227_657_679_F TGTTGGTGCTT 645 OMPA_AY485227_139_765_RTAAGCCAGCAAGA 871 TCTGGCGCTTA GCTGTATAGTTCC A A 923OMPA_AY485227_660_683_F TGGTGCTTTCT 613 OMPA_AY485227_786_807_RTACAGGAGCAGCA 884 GGCGCTTAAAC GGCTTCAAG GA 924 GYRA_AF100557_4_23_FTCTGCCCGTGT 402 GYRA_AF100557_119_142_R TCGAACCGAAGTT 1063 CGTTGGTGAACCCTGACCAT 925 GYRA_AF100557_70_94_F TCCATTGTTCG 316GYRA_AF100557_178_201_R TGCCAGCTTAGTC 1211 TATGGCTCAAG ATACGGACTTC 926GYRB_AB008700_19_40_F TCAGGTGGCT 289 GYRB_AB008700_111_140_RTATTGCGGATCAC 941 TACACGGCGT CATGATGATATTC AG TTGC 927GYRB_AB008700_265_292_F TCTTTCTTGAA 420 GYRB_AB008700_369_395_RTCGTTGAGATGGT 1113 TGCTGGTGTAC TTTTACCTTCGT GTATCG TG 928GYRB_AB008700_368_394_F TCAACGAAGGT 251 GYRB_A3008700_466_494_RTTTGTGAAACAGC 1440 AAAAACCATCT GAACATTTTCTTG CAACG GTA 929GYRB_AB008700_477_504_F TGTTCGCTGTT 641 GYRB_AB008700_611_632_RTCACGCGCATCAT 977 TCACAAACAAC CACCAGTCA ATTCCA 930GYRB_AB008700_760_787_F TACTTACTTGA 198 GYRB_AB008700_862_888_RACCTGCAATATCT 729 GAATCCACAAG AATGCACTCTTAC CTGCAA G 931WAAA_Z96925_2_29_F TCTTGCTCTTT 416 WAAA_Z96925_115_138_R CAAGCGGTTTGCC758 CGTGAGTTCAG TCAAATAGTCA TAAATG 932 WAAA_Z96925_286_311_F TCGATCTGGTT360 WAAA_Z96925_394_412_R TGGCACGAGCCTG 1274 TCATGCGTTT ACCTGT 939RPOB_EC_3798_3821_F TGGGCAGCGTT 581 RPOB_EC_9862_3889_R TGTCCGACTTGAC1326 TCGGCGAAATG GGTCAGCATTTCC GA TG 940 RPOB_EC_3798_3821_F TGGGCAGCGTT581 RPOB_EC_3862_3899_2_R TGTCCGACTTGAC 1327 TCGGCGAAATG GGTTAGCATTTCCGA TG 941 TUFB_EC_275_299_F TGATCACTGGT 468 TUFB_EC_337_362_RTGGATGTGCTCAC 1271 GCTGCTCAGAT GAGTCTGTGGCAT GGA 942 TUFB_EC_251_278_FTGCACGCCGAC 493 TUFB_EC_337360_R TATGTGCTCACGA 937 TATGTTAAGAAGTTTGCGGCAT CATGAT 949 GYRE_AB008700_760_787_F TACTTACTTGA 198GYRB_AB008700_862_888_2_R TCCTGCAATATCT 1050 GAATCCACAAG AATGCACTCTTACCTGCAA G 958 RPOC_EC_2223_2243_F TGGTATGCGTG 605 RPOC_EC_2329_2352_RTGCTAGACCTTTA 1243 GTCTGATGGC CGTGCACCGTG 959 RPOC_EC_918_938_FTCTGGATAACG 404 RPOC_EC_1009_1031_R TCCAGCAGGTTCT 1004 GTCGTCGCGGGACGGAAACG 960 RPOC_EC_2334_2357_F TGCTCGTAAGG 523 RPOC_EC_2380_2403_RTACTAGACGACGG 905 GTCTGGCGGAT GTCAGGTAACC AC 961 RPOC_EC_917_938_FTATTGGACAAC 242 RPOC_EC_1009_1034_R TTACCGAGCAGGT 1362 GGTCGTCGCGGTCTGACGGAAACG 962 RPOB_E2005_2027_F TCGTTCCTGGA 387 RPOB_EC_2041_2064_RTTGACGTTGCATG 1399 ACACGATGACG TTCGAGCCCAT C 963 RPOB_EC_1527_1549_FTCAGCTGTCGC 282 RPOB_EC_1630_1649_R TCGTCGCGGACTT 1104 AGTTCATGGACCGAAGCC C 964 INFB_EC_1347_1367_F TGCGTTTACCG 515 INFB_EC_1414_1432_RTCGGCATCACGCC 1090 CAATGCGTGC GTCGTC 965 VALS_EC_1128_1151_F TATGCTGACCG237 VALS_EC_1231_1257_R TTCGCGCATCCAG 1384 ACCAGTGGTAC GAGAAGTACATGT GTT 978 RPOC_EC_2145_2175_F TCAGGAGTCGT 285 RPOC_EC_2228_2247_RTTACGCCATCAGG 1363 TCAACTCGATC CCACGCA TACATGATG 1045CJST_CJ_1668_1700_F TGCTCGAGTGA 522 CJST_CJ_1774_1799_R TGAGCGTGTGGAA1170 TTGACTTTGCT AAGGACTTGGATG AAATTTAGAGA 1046 CJST_CJ_2171_2197_FTCGTTTGGTGG 388 CJST_CJ_2283_2313_R TCTCTTTCAAAGC 1126 TGGTAGATGAAACCATTGCTCATT AAAGG ATAGT 1047 CJST_CJ_584_616_F TCCAGGACAAA 315CJST_CJ_663_692_R TTCATTTTCTGGT 1379 TGTATGAAAAA CCAAAGTAAGCAGTGTCCAAGAAG TATC 1048 CJST_CJ_360_394_F TCCTGTTATCC 346CJST_CJ_442_476_R TCAACTGGTTCAA 955 CTGAAGTAGTT AAACATTAAGTTGAATCAAGTTTG TAATTGTCC TT 1049 CJST_CJ_2636_2668_F TGCCTAGAAGA 504CJST_CJ_2753_2777_R TTGCTGCCATAGC 1409 TCTTAAAAATT AAAGCCTACAGCTCCGCCAACTT 1050 CJST_CJ_1290_1320_F TGGCTTATCCA 575 CJST_CJ_1406_1433_RTTTGCTCATGATC 1437 AATTTAGATCG TGCATGAAGCATA TGGTTTTAC AA 1051CJST_CJ_3267_3293_F TTTGATTTTAC 707 CJST_CJ_3356_3385_R TCAAAGAACCCGC951 GCCGTCCTCCA ACCTAATTCATCA GGTCG TTTA 1052 CJST_CJ_5_39_F TAGGCGAAGAT222 CJST_CJ_104_137_R TCCCTTATTTTTC 1029 ATACAAAGAGT TTTCTACTACCTTATTAGAAGCT CGGATAAT AGA 1053 CJST_CJ_1080_1110_F TTGAGGGTATG 681CJST_CJ_1166_1198_R TCCCTCATGTTT 1022 CACCGTCTTT AAATGATCAGGATTTGATTCTTT AAAAAGC 1054 CJST_CJ_2060_2090_F TCCCGGACTTA 323CJST_CJ_2148_2174_R TCGATCCGCATCA 1068 ATATCAATGAA CCATCAAAAGCAAAATTGTGGA A 1055 CJST_CJ_2869_2895_F TGAAGCTTGTT 432 CJST_CJ_2979_3007_RTCCTCCTTGTGCC 1045 CTTTAGCAGGA TCAAAACGCATTT CTTCA TTA 1056CJST_CJ_1880_1910_F TCCCAATTAAT 317 CJST_CJ_1981_2011_R TGGTTCTTACTTG1309 TCTGCCATTTT CTTTGCATAAACT TCCAGGTAT TTCCA 1057 CJST_CJ_2185_2212_FTAGATGAAAAG 208 CJST_CJ_2283_2316_R TGAATTCTTTCAA 1152 GGCGAAGTGGCAGCACCATTGCTC TAATGG ATTATAGT 1058 CJST_CJ_1643_1670_F TTATCGTTTGT 660CJST_CJ_1724_1752_R TGCAATGTGTGCT 1198 GGAGCTAGTGC ATGTCAGCAAA TTATGCAAGAT 1059 CJST_CJ_2165_2194_F TGCGGATCGTT 511 CJST_CJ_2247_2278_RTCCACACTGGATT 1002 TGGTGGTTGTA GTAATTTACCTTG GATGAAAA TTCTTT 1060CJST_CJ_599_632_F TGAAAAATGTC 424 CJST_CJ_711_743_R TCCCGAACAATGA 1024CAAGAAGCATA GTTGTATCAACTA GCAAAAAAAGC TTTTTAC A 1061 CJST_CJ_360_393_FTCCTGTTATCC 345 CJST_CJ_443_477_R TACAACTGGTTCA 882 CTGAAGTAGTTAAAACATTAAGCT AATCAAGTTTG GTAATTGTC T 1062 CJST_CJ_2678_2703_FTCCCCAGGACA 321 CJST_CJ_2760_2787_R TGTGCTTTTTTTG 1339 CCCTGAAATTTCTGCCATAGCAAA CAAC GC 1063 CJST_CJ_1268_1299_F AGTTATAAACA 29CJST_CJ_1349_1379_R TCGGTTTAAGCTC 1096 CGGCTTTCCTA TACATGATCGTAATGGCTTATCC GGATA 1064 CJST_CJ_1680_1713_F TGATTTTGCTA 479CJST_CJ_1795_1822_R TATGTGTAGTTGA 938 AATTTAGAGAA GCTTACTACATGAATTGCGGATGA GC A 1065 CJST_CJ_2857_2887_F TGGCATTTCTT 565CJST_CJ_2965_2998_R TGCTTCAAAACGC 1253 ATGAAGCTTGT ATTTTTACATTTTTCTTTAGCA CGTTAAAG 1070 RNASEP_BKM_580_599_F TGCGGGTAGGG 512RNASEP_BKM_665_686_R TCCGATAAGCCGG 1034 AGCTTGAGC ATTCTGTGC 1071RNASEP_BKM_616_637_F TCCTAGAGGAA 333 RNASEP_BKM_665_687_R TGCCGATAAGCCC1222 TGGCTGCCACG GGATTCTGTGC 1072 RNASEP_BDP_574_592_F TGGCACGGCCA 561RNASEP_BDP_616_635_R TCGTTTCACCCTG 1115 TCTCCGTG TCATGCCG 107323S_BRM_1110_1129_F TGCGCGGAAGA 510 23S_BRM_1176_1201_R TCGCAGGCTTACA1074 TGTAACGGG GAACGCTCTCCTA 1074 23S_BRM_515_536_F TGCATACAAAC 49623S_BRM_616_635_R TCGGACTCGCTTT 1088 AGTCGGAGCCT CGCTACG 1075RNASEP_CLB_459_487_F TAAGGATAGTG 162 RNASEP_CLB_498_526_R TGCTCTTACCTCA1247 CAACAGAGATA CCGTTCCACCCTT TACCGCC ACC 1076 RNASEP_CLB_459_487_FTAAGGATAGTG 162 RNASEP_CLB_498_522_R TTTACCTCGCCTT 1426 CAACAGAGATATCCACCCTTACC TACCGCC 1077 ICD_CXB_93_120_F TCCTGACCGAC 343ICD_CXB_172_194_R TAGGATTTTTCCA 921 CCATTATTCCC CGGCGGCATC TTTATC 1078ICD_CXB_92_120_F TTCCTGACCGA 671 ICD_CXB_172_194_R TAGGATTTTTCCA 921CCCATTATTCC CGGCGGCATC CTTTATC 1079 ICD_CXB_176_198_F TCGCCGTGGAA 369ICD_CXB_224_247_R TAGCCTTTTCTCC 916 AAATCCTACGC GGCGTAGATCT T 1080IS1111A_NC002971_6866_(—) TCAGTATGTAT 290 IS1111A_NC002971_6928_6954_RTAAACGTCCGATA 848 6891_F CCACCGTAGCC CCAATGGTTCGCT GTC C 1081IS1111A_NC002971_7456_(—) TGGGTGACATT 594 IS1111A_NC002971_7529_7554_RTCAACAACACCTC 952 7483_F CATCAATTTCA CTTATTCCCACTC TCGTTC 1082RNASEP_RKP_419_448_F TGGTAAGAGCG 599 RNASEP_RKP_542_565_R TCAAGCGATCTAC957 TGGTAACA CCGCATTACAA 1083 RNASEP_RKP_422_443_F TAAGAGCGCAC 159RNASEP_RKP_542_565_R TCAAGCGATCTAC 957 CGGTAAGTTGG CCGCATTACAA 1084RNASEP_RKP_466_491_F TCCACCAAGAG 310 RNASEP_RKP_542_565_R TCAAGCGATCTAC957 CAAGATCAAAT CCGCATTACAA AGGC 1085 RNASEP_RKP_264_287_F TCTAAATGGTC391 RNASEP_RKP_295_321_R TCTATAGAGTCCG 1119 GTGCAGTTGCG GACTTTCCTCGTG TGA 1086 RNASEP_RKP_426_448_F TGCATACCGGT 497 RNASEP_RKP_542_565_RTCAAGCGATCTAC 957 AAGTTGGCAAC CCGCATTACAA A 1087 OMPB_RKP_860_890_FTTACAGGAAGT 654 OMPB_RKP_972_996_R TCCTGCAGCTCTA 1051 TTAGGTGGTAACCTGCTCCATTA TCTAAAAGG 1088 OMPB_RKP_1192_1221_F TCTACTGATTT 392OMPB_RKP_1288_1315_R TAGCAgCAAAGT 910 TGGTAATCTTG TATCACACCTGCA CAGCACAGGT 1089 OMPB_RKP_3417_3440_F TGCAAGTGGTA 485 OMPB_RKP_3520_3550_RTGGTTGTAGTTCC 1310 CTTCAACATGG TGTAGTTGTTGCA GG TTAAC 1090GLTA_RKP_1043_1072_F TGGGACTTGAA 576 GLTA_RKP_1138_1162_R TGAACATTTGCGA1147 GCTATCGCTCT CGGTATACCCAT TAAAGATG 1091 GLTA_RKP_400_428_FTCTTCTCATCC 413 GLTA_RKP_499_529_R TGGTGGGTATCTT 1305 TATGGCTATTAAGCAATCATTCTA TGCTTGC ATAGC 1092 GLTA_RKP_1023_1055_F TCCGTTCTTA 330GLTA_RKP_1129_1156_R TTGGCGACGGTAT 1415 AAATAGCAATA ACCCATAGCTTTAGAACTTGAAGC TA 1093 GLTA_RKP_1043_1072_2_F TGGAGCTTGAA 553GLTA_RKP_1138_1162_R TGAACATTGCGA 1147 GCTATCGCTCT CGGTATACCCAT AAAGATG1094 GLTA_RKP_1043_1072_3_F TGGAACTTGAA 543 GLTA_RKP_1138_1164_RTGTGAACATTTGC 1330 GCTCTCGCTCT GACGGTATACCCA TAAAGATG T 1095GLTA_RKP_400_428_F TCTTCTCATCC 413 GLTA_RKP_505_534_R TGCGATGGTAGGT 1230TATGGCTATTA ATCTTAGCAATCA TGCTTGC TTCT 1096 CTXA_VBC_117_142_FTCTTATGCCAA 410 CTXA_VBC_194_218_R TGCCTAACAAATC 1226 GAGGACAGAGTCCGTCTGAGTTC GAGT 1097 CTXA_VBC_351_377_F TGTATTAGGGG 630CTXA_VBC_441_466_R TGTCATCAAGCAC 1324 CATACAGTCCT CCAAAATGAACT CATCC1098 RNASEP_VBC_331_349_F TCCGCGGAGTT 325 RNASEP_VBC_388_414_RTGACTTTCCTCCC 1163 GACTGGGT CCTTATCAGTCTC C 1099 TOXR_VBC_135_158_FTCGATTAGGCA 362 TOXR_VBC_221_246_R TTCAAAACCTTGC 1370 GCAACGAAAGCTCTCGCCAAACAA CG 1100 ASD_FRT_1_29_F TTGCTTAAAGT 690 ASD_FRT_86_116_RTGAGATGTCGAAA 1164 TGGTTTTATTG AAAACGTTGGCAA GTTGGCG AATAC 1101ASD_FRT_43_76_F TCAGTTTTAAT 295 ASD_FRT_129_156_R TCCATATTGTTGC 1009GTCTCGTATGA ATAAAACCTGTTG TCGAATCAAAA GC G 1102 GALE_FRT_168_199_FTTATCAGCTAG 658 GALE_FRT_241_269_R TCACCTACAGCTT 973 ACCTTTTAGGTTAAAGCCAGCAAA AAAGCTAAGC ATG 1103 GALE_FRT_834_865_F TCAAAAAGCCC 245GALE_FRT_901_925_R TAGCCTTGGCAAC 915 TAGGTAAAGAG ATCAGCAAAACT ATTCCATATC1104 GALE_FRT_308_339_F TCCAAGGTACA 306 GALE_FRT_390_422_R TCTTCTGTAAAGG1136 CTAAACTTACT GTGGTTTATTATT TGAGCTAATG CATCCCA 1105IPAH_SGF_258_277_F TGAGGACCGTG 458 IPAH_SGF_301_327_R TCCTTCTGATGCC 1055TCGCGCTCA TGATGGACCAGGA G 1106 IPAH_SGF_113_134_F TCCTTGACCGC 350IPAH_SGF_172_191_R TTTTCCAGCCATG 1441 CTTTCCGATAC CAGCGAC 1107IPAH_SGF_462_486_F TCAGACCATGC 271 IPAH_SGF_522_540_R TGTCACTCCCGAC 1322TCGCAGAGAAA ACGCCA CTT 1111 RNASEP_BRM_461_488_F TAAACCCCATC 147RNASEP_BRM_542_561_R TGCCTCGCGCAAC 1227 GGGAGCAAGAC CTACCCG CGAATA 1112RNASEP_BRM_325_347_F TACCCCAGGGA 185 RNASEP_BRM_402_428_R TCTCTTACCCCAC1125 AAGTGCCACAG CCTTTCACCCTTA A C 1128 HUPB_CJ_113_134_F TAGTTGCTCAA230 HUPB_CJ_157_188_R TCCCTAATAGTAG 1028 ACAGCTGGGCT AAATAACTGCATCAGTAGC 1129 HUPB_CJ_76_102_F TCCCGGAGCTT 324 HUPB_CJ_157_188_RTCCCTAATAGTAG 1028 TTATGACTAAA AAATAACTGCATC GCAGAT AGTAGC 1130HUPB_CJ_102_F TCCCGGAGCTT 324 HUPB_CJ_114_135_R TAGCCCAGCTGTT 913TTATGACTAAA TGAGCAACT GCAGAT 1151 AB_MLST-11- TGAGATTGCTG 454AB_MLST-11- TTGTACATTTGAA 1418 OIF007_62_91_F AACATTTAATGOIF007_169_203_R ACAATATGCATGA CTGATTGA CATGTGAAT 1152 AB_MLST-11-TATTGTTTCAA 243 AB_MLST-11- TCACAGGTTCTAC 969 OIF007_185_214_FATGTACAAGGT OIF007_291_324_R TTCATCAATAATT GAAGTGCG TCCATTGC 1153AB_MLST-11- TGGAACGTTAT 541 AB_MLST-11- TTGCAATCGACAT 1400OIF007_260_289_F CAGGTGCCCCA OIF007_364_393_R ATCCATTTCACCA AAATTCG TGCC1154 AB_MLST-11- TGAAGTGCGTG 436 AB_MLST-11- TCCGCCAAAAACT 1036OIF007_206_239_F ATGATATCGAT OIF007_318_344_R CCCCTTTTCACAG GCACTTGATGTG A 1155 AB_MLST-11- TCGGTTTAGTA 378 AB_MLST-11- TTCTGCTTGAGGA 1392OIF007_522_552_F AAAGAACGTAT OIF007_587_610_R ATAGTGCGTGG TGCTCAACC 1156AB_MLST-11- TCAACCTGACT 250 AB_MLST-11- TACGTTCTACGAT 902OIF007_547_571_F GCGTGAATGGT OIF007_656_686_R TTCTTCATCAGGT TGT ACATC1157 AB_MLST-11- TCAAGCAGAAG 256 AB_MLST-11- TACAACGTGATAA 881OIF007_601_627_F CTTTGGAAGAA OIF007_710_736_R ACACGACCAGAAG GAAGG C 1158AB_MLST-11- TCGTGCCCGCA 384 AB_MLST-11- TAATGCCGGGTAG 878OIF007_1202_1225_F ATTGCATAAA OIF007_1266_1296_R TGCAATCCATTCT GC TCTAG1159 AB_MLST-11- TCGTCCCGCA 384 AB_MLST-11- TGCACCTGCGGTC 1199OIG007_1202_1225_F ATTTGCATAAA OIF007_1299_1316_R GAGCG GC 1160AB_MLST-11- TTGTAGCACAG 694 AB_MLST-11- TGCCATCCATAAT 1215OIF007_1234_1264_F TCCTGAAAC OIF007_1335_1362_R CACGCCATACTGA CG 1161AB_MLST-11- TAGGTTTACGT 225 AB_MLST-11- TGCCAGTTTCCAC 1212OIF007_1327_1356_F GATTATGG OIF007_1422_1448_R ATTTCACGTTCGT G 1162AB_MLST-11- TCGTGATTATG 383 AB_MLST-11- TCGCTTGAGTGTA 1083OIF007_1345_1369_F AA OIF007_1470_1494_R GTCATGATTGCG 1163 AB_MLST-11-TTATGGATGGC 662 AB_MLST-11- TCGCTTGAGTGTA 1083 OIF007_1351_1375_F GTOIF007_1470_1494_R GTCATGATTGCG 1164 AB_MLST-11- TCTTTGCCATT 422AB_MLST-11- TCGCTTGAGTGTA 1083 OIF007_1387_1412_F GAAGATGACTTOIF007_1470_1494_R GTCATAGATTGCG AAGC 1165 AB_MLST-11- TACTAGCGGTA 194AB_MLST-11- TGAGTCGGGTTCA 1173 OIF007_1542_1569_F AGCTTAAACAAOIF007_1656_1680_R CTTACCTGGCA GATTGC 1166 AB_MLST-11- TTGCCAATGAT 684AB_MLST-11- TGAGTCGGGTTCA 1173 OIF007_1566_1593_F ATTCGTTGGTTOIF007_1656_1680_R CTTTACCTGGCA AGCAAG 1167 AB_MLST-11- TCGGCGAAATC 375AB_MLST-11- TACCGGAAGCACC 890 OIF007_1611_1638_F CGTATTCCTGAOIF007_1731_1757_R AGCGACATTAATA AAATGA G 1168 AB_MLST-11- TACCACTATTA182 AB_MLST-11- TGCAACTGAATAG 1195 OIF007_1726_1752_F ATGTCGCTGGTOIF007_1790_1821_R ATTGCAGTAAGTT GCTTC ATAAGC 1169 AB_MLST-11-TTATAACTTAC 656 AB_MLST-11- TGAATTATGCAAG 1151 OIF007_1792_1826_FTGCAATCTATT OIF007_1876_1909_R AAGTGATCAATTT CAGTTGCTG TCTCACGA GTG 1170AB_MLST-11- TTATAACTTAC 656 AB_MLST-11- TGCCGTAACTAAC 1224OIF007_1792_1826_F TGCAATCTATT OIF007_1895_1927_R ATAAGAGAATTATCAGTTGCTTGG GCAAGAA TG 1171 AB_MLST-11- TGGTTATGTAC 618 AB_MLST-11-TGACGGCATCGA 1157 OIF007_1970_2002_F CAAATACTTTG OIF007_2097_2118_RACCACCGTC TCTGAAGAGG 1172 RNASEP_BRM_461_488_F TAAACCCCATC 147RNASEP_BRM_542_561_2_R TGCCTCGTGCAAC 1228 GGGAGCAAGAC CCACCCG CGAATA2000 CTXB_NC002505_46_70_F TCAGCGTATGC 278 CTSB_NC002505_132_162_RTCCGGCTAGAGAT 1039 ACATGGAACTC TCTGTATACGAC CTC AATATC 2001FUR_NC002505_87_113_F TGAGTGCCAAC 465 FUR_NC002505_205_228_RTCCGCCTTCAAAA 1037 ATATCAGTGCT TGGTGGCGAGT GAAGA 2002FUR_NC002505_87_113_F TGAGTGCCAAC 465 FUR_NC002505_178_205_RTCACGATACCTGC 974 ATATCAGTGCT ATCATCAAATTG GAAGA GTT 2003GAPA_NC002505_533_560_F TCGACAACACC 356 GAPA_NC002505_646_671_RTCAGAATCGATGC 980 ATTATCTATGG CAAATGCGTCATC 2004GAPA_NC002505_505_19_721_F TCAATGAACGA 259 GAPA_NC002505_769_798_RTCCTCTATGCAAC 1046 CCAACAAGTGA TTAGTATCAACA TTGATG GAAT 2005GAPA_NC002505_753_782_F TGCTAGTCAAT 517 GAPA_NC002505_856_881_RTCCATCGCAGTCA 1011 CTATCATTCCG CGTTTACTGTTGG GTTGATAC 2006GYRB_NC002505_2_32_F TGCCGGACAAT 501 GYRB_NC002505_109_134_RTCCACCACCTCAA 1003 TACGATTCATC AGACCATGTGGTG GAGTATTAA 2007GYRB_NC002505_123_152_F TGAGGTGGTGG 460 GYRB_NC002505_199_225_RTCCGTCATCGCTG 1042 ATAACTCAATT ACAGAAACTGAGT GATGAAGC T 2008GYRB_NC002505_768_794_F TATGCAGTGGA 236 GYRB_NC002505_832_860_RTGGAAACCGGCTA 1262 ACGATGGTTTC AGTGAGTACCACC CAAGA ATC 2009GYRB_NC002505_837_860_F TGGTACTCACT 603 GYRB_NC002505_937_957_RTCCTTCACGCGCA 1054 TAGCGGGTTT TCATCACC CG 2010 GYRB_NC002505_934_956_FTCGGGTGATGA 377 GYRB_NC002505_982_1007_R TGGCTTGAGAATT 1283 TGCGCGTGAAGTAGGATCCGGCAC G 2011 GYRB_NC002505_1161_1190_F TAAAGCCCGTG 148GYRB_NC002505_1255_1284_R TGAGTCACCCTCC 1172 AAATGACTCGT ACAATGTATAGTTCGTAAAGG CAGA 2012 OMPU_NC002505_275_110_F TACGCTGACGG 190OMPU_NC002505_154_180_R TGCTTCAGCACGG 1254 AATCAACCAAA CCACCAACTTCTAGCGG G 2013 OMPU_NC002505_258_283_F TGACGGCCTAT 451OMPU_NC002505_346_369_R TCCGAGACCAGCG 1033 ACGGTGTTGGT TAGGTGTAACG TTCT2014 OMPU_NC002505_431_455_F TCACCGATATC 266 OMPU_NC002505_544_567_RTCGGTCAGCAAAA 1094 ATGGCTTACCA CGGTAGCTTGC CGG 2015OMPU_NC002505_533_557_F TAGGCGTGAAA 223 OMPU_NC002505_625_651_RTAGAGAGTAGCCA 908 GCAAGCTACCG TCTTCACCGTTGT TTT C 2016OMPU_NC002505_689_713_F TAGGTGCTGGT 224 OMPU_NC002505_725_751_RTGGGGTAAGACGC 1291 TACGCAGATCA GGCTAGCATGTAT AGA T 2017OMPU_NC002505_727_747_F TACATGCTAGC 181 OMPU_NC002505_811_835_RTAGCAGCTAGCTC 911 CGCGTCTTAC GTAACCAGTGTA 2018 OMPU_NC002505_931_953_FTACTACTTCAA 193 OMPU_NC002505_1033_1053_R TTAGAAGTCGTAA 1368 GCCGAACTTCCCGTGGACC G 2019 OMPU_NC002505_927_953_F TACTTACTACT 197OMPU_NC002505_1033_1054_R TGGTTAGAAGTCG 1307 TCAAGCCGAAC TAACGTGGACCTTCCG 2020 TCPA_NC002505_48_73_F TCACGATAAGA 269 TCPA_NC002505_148_170_RTTCTGCGAATCAA 1391 AAACCGGTCAA TCGCACGCTG GAGG 2021TDH_NC004605_265_289_F TGGCTGACATC 574 TDH_NC004605_357_386_RTGTTGAAGCTGTA 1351 CTACATGACTG CTTGACCTGATTT TGA TACG 2022VVHA_NC004460_772_802_F TCTTATTCCAA 412 VVHA_NC004460_862_886_RTACCAAAGCGTGC 887 CTTCAAACCGA ACGATAGTTGAG ACTATGACG 202323S_EC_2643_2667_F TGCCTGTTCTT 508 23S_EC_2746_2770_R TGGGTTTCGCGCT 1297AGTACGAGAGG AGATGCTTTCA ACC 2024 16S_EC_713_732_TMOD_F TAGAACACCCG 20216S_EC_789_811_R TGCGTGGACTAC 1240 ATGGCGAAGGC AGGGTATCTA 202516S_EC_784_806_F TGGATTAGAGA 560 16S_EC_880_897_TMOD_R TGGCCGTACTCCC1278 CCCTGGTAGTC CAGGCG C 2026 16S_EC_959_981_F TGTCGATGCAA 63416S_EC_1052_1074_R TACGAGCTGACGA 896 CGCGAAGAACC CAGCCATGCA T 2027TUFB_EC_956_979_F TGCACACGCCG 489 TUFB_EC_1034_1058_2_R TGCATCACCATTT1204 TTCTTCAACAA CCTTGTCCTTCG CT 2028 RPOC_EC_2146_2174_TMOD_FTCAGGAGTCGT 284 RPOC_EC_2227_2249_R TGCTAGGCCATCA 1244 TCAACTCGATCGGCCACGCAT TACATGAT 2029 RPOB_EC_1841_1866_F TGGTTATCGCT 617RPOB_EC_1909_1929_TMOD_R TGCTGGATTCGCC 1250 CAGGCGAACTC TTTGCTACG CAAC2030 RPLB_EC_650_679_TMOD_F TGACCTACAGT 449 RPLB_EC_739_763_RTGCCAAGTGCTGG 1208 AAGAGGTTCTG TTTACCCCATGG AATGAACC 2031RPLB_EC_690_710_F TCCACACGGTG 309 RPLB_EC_737_760_R TGGGTGCTGGTTT 1295GTGGTGAAGG ACCCCATGGAG 2032 INFB_EC_1366_1393_F TCTCGTGGTGC 397INFB_EC_1439_1469_R TGTGCTGCTTTCG 1335 ACAAGTAACGG CATGGTTAATTGC ATATTATTCAA 2033 VALS_EC_1105_1124_TMOD_F TCGTGGCGGCG 385 VALS_EC_1195_1219_RTGGGTACGAACTG 1292 TGGTTATCGA GATGTCGCCGTT 2034 SSPE_BA_113_137_FTGCAAGCAAAC 482 SSPE_BA_197_222_TMOD_R TTGCACGTCTGTT 1402 GCACAATCAGATCAGTTGCAAATT AGC C 2035 RPOC_EC_2218_2241_TMOD_F TCTGGCAGGTA 405RPOC_EC_2313_2338_R TGGCACCGTGGGT 1273 TGCGTTGTCTG TGAGATGAAGTAC ATG2056 MECI-NC003923- TTTACACATAT 698 MECI-NC003923-41798- TTGTGATATGGAG1420 41798-41609_33_60_F CGTGAGCAATG 41609_86_113_R GTGTAGAAGGTGT AACTGATA 2057 AGR-III_NC003923- TCACCAGTTTG 263 AGR-III_NC003923-ACCTGCATCCCTA 730 2108074- CCACGTATCTT 2108074- AACGTACTTGC2109508_1_23_F CAA 2109507_56_79_R 2058 AGR-III_NC003923- TGAGCTTTTAG457 AGR-III_NC003923- TACTTCAGCTTCG 906 2108074- TTGACTTTTTC 2108074-TCCAATAAAAAAT 2109507_569_596_F AACAGC 2109507_622_653_R CACAAT 2059AGR-III_NC003923- TTTCACACAGC 701 AGR-III_NC003923- TGTAGGCAAGTGC 13192108074- GTGTTTATAGT 2108074- ATAAGAAATTGAT 2109507_1024_1052_F TCTACCA2109507_1070_1098_R ACA 2060 AGR- TGGTGACTTCA 610 AGR- TCCCCATTAATAA1021 I_AJ617706_622_651_F TAATGGATGAA I_AJ617706_694_726_R TTCCACCTACTATGTTGAAGT CACACT 2061 AGR- TGGGATTTTAA 579 AGR- TGGTACTTCAACT 1302I_AJ617706_580_611_F AAAACATTGGT I_AJ617706_626_655_R TCATCCATTATGAAACATCGCAG AGTC 2062 AGR-II_NC002745- TCTTGCAGCAG 415 AGR-II_NC002745-TTGTTTATTGTTT 1424 2079448- TTTATTTGATG 2079448- CCATATGCTACAC2080879_620_651_F AACCTAAAGT 2080879_700_731_R ACTTTC 2063AGR-II_NC002745- TGTACCCGCTG 624 AGR-II_NC002745- TCGCCATAGCTAA 10772079448- AATTAACGAAT 2079448- GTTGTTTATTGTT 2080879_649_679_F TTATACGAC2080879_715_745_R TCCAT 2064 AGR- TGGTATTCTAT 606 AGR- TGCGCTATCAACG1233 IV_AJ617711_931_961_F TTTGCTGATAA IV_AJ617711_1004_1035_RATTTTGACAATAT TGACCTCGC ATGTGA 2065 AGR- TGGCACTCTTG 562 AGR-TCCCATACCTATG 1017 IV_AJ617711_250_283_F CCTTTAATATTIV_AJ617711_309_335_R GCGATAACTGTCA AGTAAACTATC T A 2066 BLAZ_NC002952TCCACTTATCG 312 BLAZ_NC002952 TGGCCACTTTTAT 1277 (1913827 . . .CAAATGGAAAA (1913827 . . . CAGCAACCTTACA 1914672)_68_68_F TTAAGCAA1914672)_68_68_R GTC 2067 BLAZ_NC002952 TGCACTTATCG 494 BLAZ_NC002952TAGTCTTTTGGAA 926 (1913827 . . . CAAATGGAAAA (1913827 . . .CACCGTCTTTAAT 1914672)_68_68_2_F 1914672)_68_68_2_R TAAAGT 2068BLAZ_NC002952 TGATACTTCAA 467 BLAZ_NC002952 TGGAACACCGTCT 1263 (1913827. . . CGCCTGCTGCT (1913827 . . . TTAATTAAAGTAT 1914672)_68_68_3_F TTC1914672_68_68_3_F CTCC 2069 BLAZ_NC002952 TATACTTCAAC 232 BLAZ_NC002952TCTTTTCTTTGCT 1145 (1913827 . . . GCCTGCTGCTT (1913827 . . .TAATTTTCCATTT 1914672)_68_68_4_F TC 1914672)_68_68_4_R GCGAT 2070BLAZ_NC002952 TGCAATTGCTT 487 BLAZ_NC002952 TTACTTCCTTACC 1366 (1913827. . . TAGTTTTAAGT (1913827 . . . ACTTTTAGTATCT 1914672)_1_33_FGCATGTAATTC 1914672)_34_67_R AAAGCATA 2071 BLAZ_NC002952 TCCTTGCTTTA 351BLAZ_NC002952 TGGGGACTTCCTT 1289 (1913827 . . . GTTTTAAGTGC (1913827 . .. ACCACTTTTAGTA 1914672)_3_34_F ATGTAATTCAA 1914672)_40_68_R TCTAA 2072BSA-A_NC003923- TAGCGAATGTG 214 BSA-A_NC003923- TGCAAGGGAAACC 11971304065- GCTTTACTTCA 1304065- TAGAATTACAAAC 1303589_99_125_F CAATT1303589_165_193_R 2073 BSA-A_NC003923- ATCAATTTGGT 32 BSA-A_NC003923-TGCATAGGAAGG 1203 1304065- GGCCAAGAAC 1304065- TAACACCATAGTT1303589_194_218_F CTGG 1303589_253_278_R 2074 BSA-A_NC003923-TTGACTGCGGC 679 BSA-A_NC003923- TAACAACGTTACC 856 1304065- ACAACACGGAT1304065- TTCGCGATCCACT 1303589_328_349_F 1303589_388_415_R 2075BSA-A_NC003923- TGCTATGGTGT 519 BSA-A_NC003923- TGTTGTGCCGCAG 13531304065- TACCTTCCCTA 1304065- TCAAATATCTAAAT 1303589_253_278_F TGCA1303589_317_344_R 2076 BSA-B_NC003923- TAGCAACAAAT 209 BSA-B_NC003923-TGTGAAGAACTTT 1331 1917149- ATATCTGAAGC 1917149- CAAATCTGTGAAT1914156_953_982_F AGCGTACT 1914156_1011_1039_R CCA 2077 BSA-B_NC003923-TGAAAAGTATG 426 BSA-B_NC003923- TCTTCTTGAAAAA 1138 1917149- GATTTGAACAA1917149- TTGTTGTCCCGAA 1914156_1050_1081_F CTCGTGAATA1914156_1109_1136_R AC 2078 BSA-B_NC003923- TCATTATCATG 300BSA-B_NC003923- TGGACTAATAACA 1267 1917149- CGCCAATGAGT 1917149-ATGAGCTCATTGT 1914156_1260_1286_F GCAGA 1914156_1323_1353_R ACTGA 2079BSA-B_NC003923- TTTCATCTTAT 703 BSA-B_NC003923- TGAATATGTAATG 11481917149- CGAGGACCCGA 1917149- CAAACCAGTCTTT 1914156_2126_2153_F ATCGA1914156_2186_2216_R GTCAT 2080 ERMA_NC002952- TCGCTATCTTA 372ERMA_NC002952- TGAGTCTACACTT 1174 55890- TCGTTGAGAAG 55890-AGGCTTAGGATGA 56621_366_392_F GGATT 56621_487_513_R A 2081ERMA_NC002952- TAGCTATCTTA 217 ERMA_NC002952- TGAGCATTTTTAT 1167 55890-TCGTTGAGAAG 55890- ATCCATCTCCACC 56621_366_395_F GGATTTGC56621_438_465_R AT 2082 ERMA_NC002952- TGATCGTTGAG 470 ERMA_NC002952-TCTTGGCTTAGGA 1143 55890- AAGGGATTTGC 55890- TGAAAATATAGTG56621_374_402_F GAAAAGA 56621_473_504_R GTGGTA 2083 ERMA_NC002952-TGCAAAATCTG 480 ERMA_NC002952- TCAATACAGAGTC 964 55890- CAACGAGCTTT55890- TACACTTGGCTTA 56621_404_427_F GG 56621_491_520_R GGAT 2084ERMA_NC002952- TCATCCTAAGC 297 ERMA_NC002952- TGGACGATATTCA 1266 55890-CAAGTGTAGAC 55890- CGGTTTACCCACT 56621_489_516_F TCTGTA 56621_586_615_RTATA 2085 ERMA_NC002952- TATAAGTGGGT 231 ERMA_NC002952- TTGACATTTGCA1397 55890- AAACCGTGAAT 55890- TGCTTCAAAGCCT 56621_586_614_F ATCGTGT56621_640_665_R G 2086 ERMC_NC005908- TCTGAACATGA 399 ERMC_NC005908-TCCGTAGTTTTG 1041 2004- TAATATCTTTG 2004- CATAATTATGGT 2738_85_116_FAAATCGGCTC 2738_173_206_R CTATTTCAA 2087 ERMC_NC005908- TCATGATAATA 298ERMC_NC005908- TTTATGGTCTAT 1429 2004- TCTTTGAAATC 2004- TTCAATGGCAGTT2738_90_120_F GGCTCAGGA 2738_460_189_R ACGAA 2088 ERMC_NC005908-TCAGGAAAAGG 283 ERMC_NC005908- TATGGTCTATTT 936 2004- GCATTTTACCC 2004-CAATGGCAGTTAC 2738_115_139_F TTG 2738_161_187_R GA 2089 ERMC_NC005908-TAATCGTGGAA 168 ERMC_NC005908- TCAACTTCTGCC 956 2004- TACGGGTTTGC 2004-ATTAAAAGTAATG 2738_374_397_F TA 2738_325_452_R CCA 2090 ERMC_NC005908-TCTTTGAAATC 421 ERMC_NC005908- TGATGGTCTATT 1185 2004- GGCTCAGGAAA 2004-TCAATGGCAGTTA 2738_101_125_F AGG 2738_159_188_R CGAAA 2091ERMB_Y13600-625- TGTTGGGAGTA 644 ERMB_Y13600-625- TCAACAATCAGA 9531362_291_321_F TTCCTTACCAT 1362_352_380_R TAGATGTCAGACG TTAAGCACA CATG2092 ERMB_Y13600-625- TGGAAAGCCAT 536 ERMB_Y13600-625- TGCAAGAGCAAC 11961362_344_367_F GCGTCTGACAT 1362_415_437_R CCTAGTGTTCG 2093ERMB_Y13600-625- TGGATATTCAC 556 ERMB_Y13600-625- TAGGATGAAAGC 9191363_404_429_F CGAACACTAGG 1362_471_493_R ATTCCGCTGGC 2094ERMB_Y13600-625- TAAGCTGCCAG 161 ERMB_Y13600-625- TCATTCTGTGGTA 9891362_465_487_F CGGAATGCTTT 1362_521_545_R TGGCGGGTAAGTT C 2095PVLUK_NC003923- TGAGCTGCATC 456 PVLUK_NC003923- TGGAAAACTCAT 12611529595- ACTGTATTGGA 1529595- GAAATTAAAGTGA 1531285_688_713_F TAG1531285_775_804_R AAGGA 2096 PVLUK_NC003923- TGGAACAAAAT 539PVLUK_NC003923- TCATTAGGTAAAA 993 1529595- AGTCTCTCGGA 1529595-TGTCTGGACATG 1531285_1039_1068_F TTTTGACT 1531285_1095_1125_R ATCCAA2097 PVLUK_NC003923- TGAGTAACATC 461 PVLUK_NC003923- TCTCATGAAAAAG 11241529595- CATATTTCTGC 1529595- GCTCAGGAGATAC 1531285_908_936_F CATACGT1531285_950_978_R AAG 2098 PVLUK_NC003923- TCGGAATCTGA 373PVLUK_NC003923- TCACACCTGTAAG 968 1529595- TGTTCAGTTGT 1529595-TGAGAAAAAGGTT 1531285_610_633_F TT 1531285_654_682_R TGAT 2099SA442_NC003923- TGTCGGTACAC 635 SA442_NC003923- TTTCCGATCAAC 14332538576- GATATTCTTCA 2538576- GTAATGAGATTTC 25388311_35_F CGA2538831_98_124_R A 2100 SA442_NC003923- TGAAATCTCAT 427 SA442_NC003923-TCGTATGACCAGC 1098 2538576- TACGTTGCATC 2538576- TTCGGTACTACTA2538831_98_124_F G 2538831_163_188_R 2101 SA442_NC003923- TCTCATTACGT395 SA442_NC003923- TTTATGACCAGCT 1428 2538576- TGCATCGGAAA 2538576-TCGGTACTACTAA 2538831_103_126_F CA 2538831_161_187_R A 2102SA442_NC003923- TAGTACCGAAG 226 SA442_NC003923- TGATAATGAAGGG 11792538576- CTGGTCATACG 2538576- AAACCTTTTTCAC 2538831_166_188_F A G2538831_231_257_R 2103 SEA_NC003923- TGCAGGGAACA 495 SEA_NC003923-TCGATCGTGACTC 1070 2052219- GCTTTAGGCA 2052219- TCTTTATTTTCAG2051456_115_135_F 2051456_173_200_R TT 2104 SEA_NC003923- TAACTCTGATG156 SEA_NC003923- TGTAATTAACCGA 1315 2052219- TTTTTGATGGG 2052219-AGGTTCTGTAGA 2051456_572_598_F AAGGT 2051456_621_651_R GTATG 2105SEA_NC003923- TGTATGGTGGT 629 SEA_NC003923- TAACCGTTTCCA 861 2052219-GTAACGTTACA 2052219- AGGTACTGTATTT 2051456_382_414_F TGATAATAAT2051456_464_492_R TGT C 2106 SEA_NC003923- TTGTATGTATG 695 SEA_NC003923-TAACCGTTTCCAA 862 2052219- GTGGTGTAACG 2052219- AGGTACTGTATTT2051456_377_406_F TTACATGA 2051456_459_492_R TGTTTACC 2107 SEB_NC002758-TTTCACATGTA 702 SEB_NC002758- TCATCTGGTTTAG 988 2135540- ATTTTGATATT2135540- GATCTGGTTGACT 2135140_208_137_F CGCACTGA 2135140_273_298_R 2108SEB_NC002758- TATTTCACATG 244 SEB_NC002758- TGCAACTCATCTG 1194 2135540-TAATTTTGATA 2135540- GTTAGGATCT 2135140_106_235_F TTCGCACT2135140_281_304_R 2109 SEB_NC002758- TAACAACTCGC 151 SEB_NC002758-TGTGCAGGCATCA 1334 2135540- CTTATGAAACG 2135540- TGTCATACCAA2135140_402_402_F GGATATA 2135140_402_402_R 2110 SEB_NC002758-TTGTATGTATG 696 SEB_NC002758- TTACCATCTTCAA 1361 2135540- GTGGTGTAACT2135540- ATACCCGAACAG 2135140_402_402_2_F GAGCA 2135140_402_402_2_R 2111SEC_NC003923- TTAACATGAAG 648 SEC_NC003923- TGAGTTTGCACTT 1177 851678-GAAACCACTTT 851678- CAAAAGAAATTGT 852768_546_575_F GATAATGG852768_620_647_R GT 2112 SEC_NC003923- TGGAATAACAA 546 SEC_NC003923-TCAGTTTGCACTT 985 851678- AACATGAAGGA 851678- CAAAAGAAATTGT852768_537_566_F AACCACTT 852768_619_647_R GTT 2113 SEC_NC003923-TGAGTTTAACA 466 SEC_NC003923- TCGCCTGGTGCAG 1078 851678- GTTCACCATAT851678- GCATCATAT 852768_720_749_F GAAACAGG 852768_794_815_R 2114SEC_NC003923- TGGTATGATAT 604 SEC_NC003923- TCTTCACACTTTT 1133 851678-GATGCCTGCAC 851678- AGAATCAACCGTT 852768_787_810_F CA 852768_853_886_RTTATTGTC 2115 SED_M28521_657_ TGGTGGTGAAA 615 SED_M28521_ TGTACACCATTTA1318 682_F TAGATAGGACT 741_770_R TCCACAAATTGAT GCTT TGGT 2116SED_M28521_ TGGAGGTGTCA 554 SED_M28521_ TGGGCACCATTTA 1288 690_711_FACTCCACACGA 739_770_R TCCACAAATTGAT A TGGTAT 2117 SED_M28521_TTGCACAAGCA 683 SED_M28521_ TCGCGCTGTATTT 1079 833_854_F AGGCGCTATTT888_911_R TTCCTCCGAGA 2118 SED_M28521_ TGGATGTTAAG 559 SED_M28521_TGTCAATATGAAG 1320 962_987_F GGTGATTTTCC 1022_1048_R GTGCTCTGTGGAT CGAAA 2119 SEA-SEE_NC002952- TTTACACTACT 699 SEA-SEE_NC002952- TCATTTATTTCTT994 2131289- TTTATTCATTG 2131289- CGCTTTTCTGCT 2130703_16_45_F CCCTAACG2130703_71_98_R AC 2120 SEA-SEE_NC002952- TGATCATCCGT 469SEA-SEE_NC002952- TAAGCACCATATA 870 2131289- GGTATAACGAT 2131289-AGTCTACTTTTTC 2130703_249_278_F TTATTAGT 2130703_314_344_R CCCTT 2121SEE_NC002952- TGACATGATAA 445 SEE_NC002952- TCTATAGGTACT 1120 2131289-TAACCGATTGA 2131289- GTAGTTTGTTTTC 2130703_409_437_F CCGAAGA2130703_485_494_R CGTCT 2122 SEE_NC002952- TGTTCAAGAGC 640 SEE_NC002952-TTTGCACCTTAC 1436 2131289- TAGATCTTCAG 2131289- CGCCAAAGCT2130703_525_550_F GCAA 2130703588_588_R 2123 SEE_NC002952- TGTTCAAGAGC639 SEE_NC002952- TACCTTACCGCC 892 2131289- TAGATCTTCAG 2131289-AAAGCTGTCT 2130703_525_549_F GCA 2130703_588_586_2_R 2124 SEE_NC002952-TCTGGAGGCAC 403 SEE_NC002952- TCCGTCTATCCA 1043 2131289- ACCAAATAAAA2131289- CAAGTTAATTGGT 2130703_381_384_F CA 2130703_444_471_R ACT 2125SEG_NC002758- TGCTCAACCCG 520 SEG_NC002758- TAACTCCTCTTCC 863 1955100-ATCCTAAATTA 1955100- TTCAACAGGTGGA 1954171_225_251_F GACGA1954171_321_348_R 2126 SEG_NC002758- TGGACAATAGA 548 SEG_NC002758-TGCTTTGTAATC 1260 1955100- CAATCACCTTG 1955100- AGTTCCTGAATAG1954171_623_851_F GATTTACA 1954171_871_702_R TAACCA 2127 SEG_NC002758-TGGAGGTTGTT 555 SEG_NC002758- TGTCTATTGTCGA 1329 1955100- GTATGTATGG1955100- ATTGTTACCTGTA 1954171_540_584_F TGTT 1954171_607_635_R CAGT2128 SEG_NC002758- TACAAAGCAAG 173 SEG_NC002758- TGATTCAAATGCA 11871955100- ACACTGGCTC 1955100- GAACCATCAAACT 1954171_694_718_F ACTA1954171_735_782_R CG 2129 SEH_NC002953- TTGCAACTGCT 682 SEH_NC002953-TAGTGTTGTACCT 927 60024- GATTTAGCTCA 60024- CCATATAGACATT60977_449_472_F GA 60977_547_578_R CAGA 2130 SEH_NC002953- TAGAAATCAAG201 SEH_NC002953- TTCTGAGCTAAAT 1390 60024- GTGATAGTGGC 60024-ACAGCAGTTGC 60977_408_434_F AAATGA 60977_450_473_R 2131 SEH_NC002953-TCTGAARTGTC 400 SEH_NC002953- TACCATCTACCC 888 60024- TATATGGAGGT 60024-AACATTAGCACCA 60977_547_576_F ACAACACTA 60977_608_634_R A 2132SEH_NC002953- TTCTGAATGTC 677 SEH_NC002953- TAGCACCAATCAC 909 60024-TATATGGAGGT 60024- CCTTTCCTGT 60977_546_575_F ACAACACT 60977_594_616_R2133 SEI_NC002758- TCAACTCGAAT 253 SEI_NC002758- TCACAAGGACCAT 9661957830- TTTCAACAGGT 1957830- TATAATCAATGCC 1956949_324_349_F ACCA1956949_419_448_R AA 2134 SEI_NC002758- TTCAACAGGTA 666 SEI_NC002758-TGTACAAGGACCA 1316 1957830- CCAATGATTTG 1957830- TTATAATCAATGC1956949_336_363_F ATCTCA 1958949_420_447_R CA 2135 SEI_NC002758-TGATCTCAGAA 471 SEI_NC002758- TCTGGCCCCTCCA 1129 1957830- TCTAATAATTG1957830- TACATGTATTTAG 1956949_356_384_F GGACGAA 1958949_449_474_R 2136SEI_NC002758- TCTCAAGGTGA 394 SEI_NC002758- TGGGTAGGTTTTT 1293 1957830-TATTGGTGTAG 1957830- ATCTGTGACGCCT 1956949_223_253_F GTAACTTAA1958949_290_316_R T 2137 SEJ_AF053140- TGTGGAGTAAC 637 SEJ_AF053140-TCTAGCGGAACAA 1118 1307_1332_F ACTGCATGAAA 1381_1404_R CAGTTCTGATG ACAA2138 SEJ_AF053140- TAGCATCAGAA 211 SEJ_AF053140- TCCTGAAGATCTA 10491378_1403_F CTGTTGTTCCG 1429_1458_R GTTCTTGAATGGT CTAG TACT 2139SEJ_AF053140- TAACCATTCAA 153 SEJ_AF053140- TAGTCCTTTCTGA 9251431_1459_F GAACTAGATCT 1500_1531_R ATTTACCATCAA TCAGGCA AGGTAC 2140SEJ_AF053140- TCATTCAAGAA 301 SEJ_AF053140- TCAGGTATGAAAC 9841434_1461_F CTAGATCTTCA 1521_1549_R ACGATTAGTCCTT GGCAAG TCT 2141TSST_NC002758- TGGTTTAGATA 619 TSST_NC002758- TGTAAAAGCAGGG 13122137564- ATTCTTTAGGA 2137564- CTATAATAAGGAC 2138293_206_236_F TCTATGCGT2138293_278_305_R TC 2142 TSST_NC002758- TGCGTATAAAA 514 TSST_NC002758-TGCCCTTTTGTAA 1221 2137564- AACACAGATGG 2137564- AAGCAGGGCTAT2138293_232_258_F CAGCA 2138293_289_313_R 2143 TSST_NC002758-TCCAAATAAGT 304 TSST_NC002758- TACTTTAAGGGGC 907 2137564- GGCGTTACAAA2137564- TATCTTTACCATG 2138293_382_410_F TACTGAAA 2138293_448_478_RAACCT 2144 TSST_NC002758- TCTTTTACAAA 423 TSST_NC002758- TAAGTTCCTTCGC874 2137564- AGGGGAAAAG 2137564- TAGTATGTTGGCT 2138293_297_325_F TTGACTT2138293_347_373_R T 2145 ARCC_NC003923- TCGCCGGCAAT 368 ARCC_NC003923-TGAGTTAAAATGC 1175 2725050- GCCATTGGATA 2725050- GATTGATTTCAGT2724595_37_58_F 2724595_97_128_R 2146 ARCC_NC003923- TGAATAGTGAT 437ARCC_NC003923- TCTTCTTCTTCG 1137 2725050- AGAACTGTAGG 2725050-TATAAAAAGGACC 2724595_131_161_F CACAATCGT 2724595_214_245_R AATTGG 2147ARCC_NC003923- TTGGTCCTTTT 691 ARCC_NC003923- TGGTGTTCTAGTA 13062725050- TATACGAAAGA 2725050- TAGATTGAGGTAG 2724595_218_249_F AGAAGTTGAA2724595_322_353_R TGGTGA 2148 AROE_NC003923- TTGCGAATAGA 686AROE_NC003923- TCGAATTCAGCTA 1064 1674726- ACGATGGCTCG 1674726-AATACTTTTCAGC 1674277_371_393_F T 1674277_435_464_R ATCT 2149AROE_NC003923- TGGGGCTTTAA 590 AROE_NC003923- TACCTGCATTAAT 891 1674726-ATATTCCAATT 1674726- CGCTTGTTCATCA 1674277_30_62_F GAAGATTTTCA1674277_155_181_R A 2150 AROE_NC003923- TGATGGCAAGT 474 AROE_NC003923-TAAGCAATACCTT 869 1674726- GGATAGGGTAT 1674726- TACTTGCACCACC1674277_204_232_F AATACAG 1674277_308_335_R TTG 2151 GLPF_NC003923-TGCACCGGCTA 491 GLPF_NC003923- TGCAACAATTAAT 1193 1296927- TTAAGAATTAC1296927- GCTCCGACAATTA 297391_270_301_F TTTGCCAACT 1297391_382_414_RAAGGATT 2152 GLPF_NC003923- TGGATGGGGAT 558 GLPF_NC003923- TAAAGACACCGCT850 1296927- TAGCGGTTACA 1296927- GGGTTTAAATGTG 1297391_27_51_F ATG1297391_81_108_R CA 2153 GLPF_NC003923- TAGCTGGCGCG 218 GLPF_NC003923-TCACCGATAAATA 972 1296927- AAATTAGGTGT 1296927- AAATACCTAAAGT1297391_239_260_F 1297391_323_359_R TAATGCCATTG 2154 GMK_NC003923-TACTTTTTTAA 200 GMK_NC003923- TGATATTGAACTG 1180 1190906- AACTAGGGATG1190906- GTGTACCATAATA 119334_91_122_F CGTTTGAAGC 1191334_166_197_RGTTGCC 2155 GMK_NC003923- TGAAGTAGAAG 435 GMK_NC003923- TCGCTCTCTCAAG1082 1190906- GTGCAAAGCAA 1190906- TGATCTAAACTTG 1191334_240_267_FGTTAGA 1191334_305_333_R GAG 2156 GMK_NC003923- TCACCTCCAAG 268GMK_NC003923- TGGGACGTAATCG 1284 1190906- TTTAGATCACT 1190906-TATAAATTCATCA 1191334_301_329_F TGAGAGA 1191334_403_432_R TTTC 2157PTA_NC003923- TCTTGTTTATG 418 PTA_NC003923- TGGTACACCTGGT 1301 628885-CTGGTAAAGCA 628885- TTCGTTTTGATGA 629355_237_263_F GATGG629355_314_345_R TTTGTA 2158 PTA_NC003923- TGAATTAGTTC 439 PTA_NC003923-TGCATTGTACCGA 1207 628885- AATCATTTGTT 628885- AGTAGTTCACATT629355_141_171_F GAACGACGT 629355_211_239_R GTT 2159 PTA_NC003923-TCCAAACCAGG 303 PTA_NC003923- TGTTCTGGATTGA 1349 628885- TGTATCAAGAA628885- TTGCACAATCACC 629355_328_356_F CATCAGG 629355_393_422_R AAAG2160 TPI_NC003923- TGCAAGTTAAG 486 TPI_NC003923- TGAGATGTTGATG 1165830671- AAAGCTGTTGC 830671- ATTACCAGTTCC 831072_131_160_F AGGTTTAT831072_209_239_R GATTG 2161 TPI_NC003923- TCCCACGAAAC 318 TPI_NC003923-TGGTACAACATCG 1300 830671- AGATGAAGAAA 830671- TTAGCTTTACCAC831072_1_34_F TTAACAAAAAA 831072_97_129_R TTTCACG G 2162 TPI_NC003923-TCAAACTGGGC 246 TPI_NC003923- TGGCAGCAATAGT 1275 830671- AATCGGAACTG830671- TTGACGTACAAAT 831072_199_227_F GTAAATC 831072_253_286_R GCACACAT2163 YQI_NC003923- TGAATTGCTGC 440 YQI_NC003923- TCGCCAGCTAGCA 1076378916- TATGAAAGGTG 378916- CGATGTCATTTTC 379431_142_167_F GCTT379431_259_284_R 2164 YQI_NC003923- TACAACATATT 175 YQI_NC003923-TTCGTGCTGGATT 1388 378916- ATTAAAGAGAC 378916- TTGTCCTTGTCCT379431_44_77_F GGGTTTGAATC 379431_120_145_R C 2165 YQI_NC003923-TCCAGCACGAA 314 YQI_NC003923- TCCAACCCAGAAC 997 378916- TTGCTGCTATG378916- CACATACTTTATT 379431_135_160_F AAAG 379431_193_221_R CAC 2166YQI_NC003923- TAGCTGGCGGT 219 YQI_NC003923- TCCATCTGTTAAA 1013 378916-ATGGAGAATAT 378916- CCATCATATACCA 379431_275_300_F GTCT 379431_364_396_RTGCTATC 2167 BLAZ_ TCCACTTATCG 312 BLAZ_ TGGCCACTTTTAT 1277 (1913827 . .. CAAATGGAAAA (1913827 . . . CAGCAACCTTACA 1914672)_546_ TTAAGCAA1914672)_655_ GTC 575_F 683_R 2168 BLAZ_ TGCACTTATCG 494 BLAZ_TAGTCTTTTGGAA 926 (1913827 . . . CAAATGGAAAA (1913827 . . .CACCGTCTTTAAT 1914672)_546_ TTAAGCAA 1914672)_628_ TAAAGT 575_2_F 659_R2169 BLAZ_ TGATACTTCAA 467 BLAZ_ TGGAACACCGTCT 1263 (1913827 . . .CGCCTGCTGCT (1913827 . . . TTAATTAAAGTAT 1914672)_507_ TTC 1914672)_622_CTCC 531_F 651_R 2170 BLAZ_ TATACTTCAAC 232 BLAZ_ TCTTTTCTTGCT 1145(1913827 . . . GCCTGCTGCTT 1913827 . . . TAATTTTCCATTT 1914672)_508_ TC1914672)_553_ GCGAT 531_F 583_R 2171 BLAZ_ TGCAATTGCTT 487 BLAZ_TTACTTCCTTACC 1366 (1913827 . . . TAGTTTTAAGT (1913827 . . .CTTTTAGTATCT 1914672)_24_ GCATGTAATTC 1914672)_121_ AAAGCATA 56_F 154_R2172 BLAZ_ TCCTTGCTTTA 351 BLAZ_ TGGGGACTTCCTT 1289 (1913827 . . .1914672)_ GTTTTAAGTGC (1913827 . . . 1914672)_ ACCACTTTTAGTA 26_58_FATGTAATTCAA 127_157_R TCTAA 2173 BLAZ_NC002952- TCCACTTATCG 312BLAZ_NC002952- TGGCCACTTTTAT 1277 1913827- CAAATGGAAAA 1913827-CAGCAACCTTACA 1914672_546_575_F TTAAGCAA 1914672_655_683_R GTC 2174BLAZ_NC002952- TGCACTTATCG 494 BLAZ_NC002952- TAGTCTTTTGGAA 926 1913827-CAAATGGAAAA 1913827- CACCGTCTTTAAT 1914672_546_575_2_F TTAAGCAA1914672_628_659_R TAAAGT 2175 BLAZ_NC002952- TGATACTTCAA 467BLAZ_NC002952- TGGAACACCGTCT 1263 1913827- CGCCTGCTGCT 1913827-TTAATTAAAGTAT 1914672_507_531_F TTC 1914672_622_651_R CTCC 2176BLAZ_NC002952- TATACTTCAAC 232 BLAZ_NC002952- TCTTTTCTTTGCT 11451913827- GCCTGCTGCTT 1913827- TAATTTTCCATTT 1914672_508_531_F TC1914672_553_583_R GCGAT 2177 BLAZ_NC002952- TGCAATTGCTT 487BLAZ_NC002952- TTACTTCCTTACC 136 1913827- TAGTTTTAAGT 1913827-ACTTTTAGTATCT 1914672_24_56_F GCATGTAATTC 1914672_121_154_R AAAGCATA2178 BLAZ_NC002952- TCCTTGCTTTA 351 BLAZ_NC002952- TGGGGACTTCCTT 12891913827- GTTTTAAGTGC 1913827- ACCACTTTTAGTA 1914672_26_58_F ATGTAATTCAA1914672_127_157_R TCTAA 2247 TUFB_NC002758- TGTTGAACGTG 643TUFB_NC002758- TGTCACCAGCTTC 1321 615038- GTCAAATCAAA 615038-AGCGTAGTCTAA 616222_693_721_F GTTGGTG 616222_793_820_R TAA 2248TUFB_NC002758- TCGTGTTGAAC 386 TUFB_NC002758- TGTCACCAGCTTC 1321 615038-GTGGTCAAATC 615038- AGCGTAGTCTAAT 616222_690_716_F AAAGT616222_793_820_R AA 2249 TUFB_NC002758- TGAACGTGGTC 430 TUFB_NC002758-TGTCACCAGCTTC 1321 615038- AAATCAAAGTT 615038- AGCGTAGTCTAAT616222_696_725_F GGTGAAGA 616222_793_820_R AA 2250 TUFB_NC002758-TCCCAGGTGAC 320 TUFB_NC002758- TGGTTTGTCAGAA 1311 615038- GATGTACCTGT615038- TCACGTTCTGGAG 616222_488_513_F AATC 616222_601_630_R TTGG 2251TUFB_NC002758- TGAAGGTGGAC 433 TUFB_NC002758- TAGGCATAACCAT 922 615038-GTCACACTCCA 615038- TTCAGTACCTTCT 616222_945_972_F TTCTTC616222_1030_1060_R GGTAA 2252 TUFB_NC002758- TCCAATGCCAC 307TUFB_NC002758- TTCCATTTCAACT 1382 615038- AAACTCGTAA 615038-AATTCTAATAATT 616222_333_356_F CA 616222_424_459_R CTTCATCGTC 2253NUC_NC002758- TCCTGAAGCAA 342 NUC_NC002758- TACGCTAAGCCAC 899 894288-GTGCATTTTAC 894288- GTCCATATTTATC 894974_402_424_F GA 894974_483_509_R A2254 NUC_NC002758- TCCTTATAGGG 349 NUC_NC002758- TGTTTGTGATGCA 1354894288- ATGGCTATCAG 894288- TTTGCTGAGCTA 894974_53_81_F TAATGTT894974_165_189_R 2255 NUC_NC002758- TCAGCAAATGC 273 NUC_NC002758-TAGTTGAAGTTGC 928 894288- ATCACAAACAG 894288- ACTATATACTGTT894974_169_194_F ATAA 894974_222_250_R GGA 2256 NUC_NC002758-TACAAAGGTCA 174 NUC_NC002758- TAAATGCACTTGC 853 894288- ACCAATGACAT894288- TTCAGGGCCATAT 894974_316_345_F TCAGACTA 89474_396_421_R 2270RPOB_EC_3798_3821_1_F TGGCCAGCGCT 566 RPOB_EC_3868_3895_R TCACGTCGTCCGA979 TCGGTGAAATG CTTCACGGGTCAG GA CT 2271 RPOB_EC_3789_3812_F TCAGTTCGGCG294 RPOB_EC_3860_3890_R TCGTCGGACTTAA 1107 GTCAGCGCTTC CGGTCAGCATTTC GGCTGCA 2272 RPOB_EC_3789_3812_F TCAGTTCGGCG 294 RPOB_EC_3860_3890_2_RTCGTCCGACTTAA 1102 GTCAGCGCTTC CGGTCAGCATTTC GG CTGCA 2273RPOB_EC_3789_3812_F TCAGTTCGGCG 294 RPOB_EC_3862_3890_R TCGTCGGACTTAA1106 GTCAGCGCTTC CGGTCAGCATTTC GG CTG 2274 RPOB_EC_3789_3812_FTCAGTTCGGCG 294 RPOB_EC_3862_3890_R TCGTCCGACTTAA 1101 GTCAGCGCTTCCGGTCAGCATTTC GG CTG 2275 RPOB_EC_3793_3812_F TTCGGCGGTCA 674RPOB_EC_3865_3890_R TCGTCGGACTTAA 1105 GCGCTTCGG CGGTCAGCATTTC 2276RPOB_EC_3793_3812_F TTCGGCGGTCA 674 RPOB_EC_3865_3890_R TCGTCCGACTTAA1100 GCGCTTCGG CGGTCAGCATTTC 2309 MUPR_X75439_1658_1689_F TCCTTTGATAT352 MUPR_X75439_1744_1773_R TCCCTTCCTTAT 1030 ATTATGCGATG ATGAGAAGGAAACGAAGGTTGGT CACT 2310 MUPR_X75439_1330_1353_F TTCCTCCTTTT 669MUPR_X75439_1413_1441_R TGAGCTGGTGCTA 1171 GAAAGCGACGG TATGAACAATACC TTAGT 2312 MUPR_X75439_1314_1338_F TTTCCTCCTTT 704 MUPR_X75439_1381_1409_RTATATGAACAATA 931 TGAAAGCGAC CCAGTTCCTTCTG GGTT AGT 2313MUPR_X75439_2486_2516_F TAATTGGGCTC 172 MUPR_X75439_2548_2574_RTTAATCTGGCGT 1360 TTTCTCGCTTA GGAAGTGAAATCG AACACCTTA T 2314MUPR_X75439_2547_2572_F TACGATTTCAC 188 MUPR_X75439_2605_2630_RTCGTCCTCTCGAA 1103 TTCCGCAGCCA TCTCCGATATACC GATT 2315MUPR_X75439_2666_2696_F TGCGTACAATA 513 MUPR_X75439_2711_2740_RTCAGATATAAATG 981 CGCTTTATGAA GAACAAATGGAGC ATTTTAACA CACT 2316MUPR_X75439_2813_2843_F TAATCAAGCAT 165 MUPR_X75439_2867_2890_RTCTGCATTTTTGC 1127 TGGAAGATGAA GAGCCTGTCTA ATGCATACC 2317MUPR_X75439_884_914_F TGACATGGACT 447 MUPR_X75439_977_1007_RTGTACAATAAGGA 1317 CCCCCTATATA GTCACCTTATGTC ACTCTTGAG CCTTA 2318CTXA_NC002505- TGGTCTTATGC 608 CTXA_NC002505- TCGTGCCTAACAA 11091568114- CAAGAGGACAG 1568114- ATCCCGTCTGAGT 1567341_114_142_F AGTGAGT1567341_194_221_R TC 2319 CTXA_NC002505- TCTTATGCCAA 411 CTXA_NC002505-TCGTGCCTAACAA 1109 1568114- GAGGACAGAGT 1568114- ATCCCGTCTGAGT1567341_117_145_F GAGTACT 1567341_194_221_R TC 2320 CTXA_NC002505-TGGTCTTATGC 608 CTXA_NC002505- TAACAAATCCCGT 855 1568114- CAAGAGGACAG1568114- CTGAGTTCCTCTT 1567341_114_142_F AGTGAGT 1567341_186_214_R GCA2321 CTXA_NC002505- TCTTATGCCAA 411 CTXA_NC002505- TAACAAATCCGT 8551568114- GAGGACAGAGT 1568114- CTGAGTTCCTCTT 1567341_117_145_F GAGTACT1567341_186_214_R GCA 2322 CTXA_NC002505- AGGACAGAGTG 27 CTXA_NC002505-TCCCGTCTGAGTT 1027 1568114- AGTACTTTGAC 1568114- CCTCTTGCATGAT1567341_129_156_F CGAGGT 1567341_180_207_R CA 2323 CTXA_NC002505-TGCCAAGAGGA 500 CTXA_NC002505- TAACAAATCCCGT 855 1568114- CAGAGTGAGTA1568114- CTGAGTTCCTCTT 1567341_122_149_F CTTTGA 1567341_186_214_R GCA2324 INV_U22457-74- TGCTTATTTAC 530 INV_U22457-74- TGACCCAAAGCT 11543772_831_858_F CTGCACTCCCA 3772_942_966_R AAAGCTTTACTG CAACTG 2325INV_U22457-74- TGAATGCTTAT 438 INV_U22457-74- TAACTGACCCAAA 8643772_827_857_F TTACCTGCACT 3772_942970_R GCTGAAAGCTTTA CCCACAACT CTG2326 INV_U22457-74- TGCTGGTAACA 526 INV_U22457-74- TGGGTTGCGTTG 12963772_1555_1581_F GAGCCTTATAG 3772_1619_1647_R AGATTATCTTTAC GCGCA CAA2327 INV_U22457-74- TGGTAACAGAG 598 INV_U22457-74- TCATAAGGGTTG 9873772_1558_1585_F CCTTATAGGCG 3772_1622_1652_R GTTGCAGATTATC CATATG TTTAC2328 ASD_NC006570- TGAGGGTTTTA 459 ASD_NC006570- TGATTCGATCATA 1188439714- TGCTTAAAGTT 439714- CGAGACATTAAA 438608_3_37_F GGTTTTATTGG438608_54_84_R CTGAGT TT 2329 ASD_NC006570- TAAAGTTGGTT 149ASD_NC006570- TCAAAATCTTTTG 948 439714- TTATTGGTTGG 439714-ATTCGATCATACG 438608_18_45_F CGCGGA 438608_66_95_R AGAC 2330ASD_NC006570- TTAAAGTTGGT 647 ASD_NC006570- TCCCAATCTTTTG 1016 439714-TTTATTGGTTG 439714- ATTCGATCATACG 438608_17_45_F GCGCGGA 438608_67_95_RAGA 2331 ASD_NC006570- TTTTATGCTTA 709 ASD_NC006570- TCTGCCTGAGATG 1128439714- AAGTTGGTTTT 439714- TCGAAAAAACGT 438608_9_40_F ATTGGTTGGC438608_107_134_R TG 2332 GALE_AF513299_171_200_F TCAGCTAGACC 280GALE_AF513299_241_271_R TCTCACCTACAGC 1122 TTTTAGGTAAA TTTAAAGCCAGCAGCTAAGCT AAATG 2333 GALE_AF513299_168_199_F TTATCAGCTAG 658GALE_AF513299_245_271_R TCTCACCTACAG 1121 ACCTTTTAGGT TTTAAAGCCAGCAAAAGCTAAGC A 2334 GALE_AF513299_168_199_F TTATCAGCTAG 658GALE_AF513299_233_264_R TACAGCTTTAAAG 883 ACCTTTTAGGT CCAGCAAAATGAAAAAGCTAAGC TTACAG 2335 GALE_AF513299_169_198_F TCCCAGCTAGA 319GALE_AF513299_252_279_R TTCAACACTCTCA 1374 CCTTTTAGGTA CCTACAGCTTTAAAAGCTAAG AG 2236 PLA_AF053945_7371_7403_F TTGAGAAGACA 680PLA_AF053945_7434_7468_R TACGTATGTAAAT 900 TCCGGCTCACG TCCGCAAAGACTTTTATTATGGTA TGGCATTAG 2337 PLA_AF053945_7377_7403_F TGACATCCGGC 443PLA_AF053945_7428_7455_R TCCGCAAAGACTT 1035 TCACGTTATTA TGGCATTAGGTGTTGGTA GA 2338 PLA_AF053945_7377_7404_F TGACATCCGGC 444PLA_AF053945_7430_7460_R TAAATTCCGCAAA 854 TCACGTTATTA GACTTTGGCATTATGGTAC GGTGT 2339 CAF_AF053947_33412_33441_F TCCGTTATCGC 329CAF_AF053947_33498_33523_R TAAGAGTGATGC 866 CATTGCATTAT GGCTGGTTCAACATTGGAACT 2340 CAF_AF053947_33426_33458_F TGCATTATTTG 499CAF_AF053947_33483_33507_R TGGTTCAACAAG 1308 GAACTATTGCA GTTGCCGTTGCAACTGCTAATGC 2341 CAF_AF053947_33407_33429_F TCAGTTCCGTT 291CAF_AF053947_33483_33504_R TTCAACAAGAGTT 1373 ATCGCCATTGC GCCGTTGCA A2342 CAF_AF053947_33407_33431_F TCAGTTCCGTT 293CAF_AF053947_33494_33517_R TGATGCGGGCTGG 1184 ATCGCCATTG TTCAACAAGAGCATT 2344 GAPA_NC_002505_1_28_F_1 TCAATGAACGA 260GAPA_NC_002505_29_58_R_1 TCCTTTATGCAAC 1060 TCAACAAGTGA TTGGTATCAACAGTTGATG GAAT 2472 OMPA_NC000117_68_89_F TGCCTGTAGGG 507OMPA_NC000117_145_167_R TCACACCAAGTAG 967 AATCCTGCTGA TGCAAGGATC A 2473OMPA_NC000117_798_821_F TGATTACCATG 475 OMPA_NC000117_865_893_RTCAAAACTTGCTC 947 AGTGGCAAGCA TAGACCATTTAAC AG TCC 2474OMPA_NC000117_645_671_F TGCTCAATCTA 521 OMPA_NC000117_757_777_RTGTCGCAGCATCT 1328 AACCTAAAGTC GTTCCTGC GAAGA 2475OMPA_NC000117_947_973_F TAACTGCATGG 157 OMPA_NC000117_1011_1040_RTGACAGGACACAA 1153 AACCCTTCTTT TCTGCATGAAGTC ACTAG TGAG 2476OMPA_NC000117_774_795_F TACTGGAACAA 196 OMPA_NC000117_871_894_RTTCAAAAGTTGCT 1371 AGTCTGCGACC CGAGACCATTG 2477 OMPA_NC000117_457_483_FTTCTATCTCGT 676 OMPA_NC000117_511_534_R TAAAGAGACGTTT 851 TGGTTTATTCGGGTAGTTCATTTG GAGTT C 2478 OMPA_NC000117_687_710_F TAGCCCAGCAC 212OMPA_NC000117_787_816_R TTGCCATTCATGG 1406 AATTTGTGATT TATTTAAGTGTAG CAAGA 2479 OMPA_NC000117_540_566_F TGGCGTAGTAG 571 OMPA_NC000117_649_672_RTTCTTGAACGCGA 1395 AGCTATTTACA GGTTTCGATTG GACAC 2480OMPA_NC000117_338_360_F TGCACGATGCG 492 OMPA_NC000117_417_444_RTCCTTTAAAATAA 1058 GAATGGTTCAC CCGCTAGTAGCTC A CT 2481OMP2_NC000117_18_40_F TATGACCAAAC 234 OMP2_NC000117_71_91_RTCCCGCTGGCAAA 1025 TCATCAGACGA TAAACTCG G 2482 OMP2_NC000117_354_382_FTGCTACGGTAG 516 OMP2_NC000117_445_471_R TGGATCACTGCTT 1270 GATCTCCTTATACGAACTCAGCTT CCTATTG C 2483 OMP2_NC000117_1297_1319_F TGGAAAGGTGT 537OMP2_NC000117_1396_1419_R TACGTTTGTATCT 903 TGCAGCTACTC TCTGCAGAACC A2484 OMP2_NC000117_1465_1493_F TCTGGTCCAAC 407 OMP2_NC000117_1541_1569_RTCCTTTCAATGTT 1062 AAAAGGAACGA ACAGAAAACTCTA TTACAGG CAG 2485OMP2_NC000117_44_66_F TGACGATCTTC 450 OMP2_NC000117_120_148_RTGTCAGCTAAGC 1323 GCGGTGACTAG TAATAACGTTTGT T AGAG 2486OMP2_NC000117_166_190_F TGACAGCGAAG 441 OMP2_NC000117_240_261_RTTGACATCGTCCC 1396 AAGGTTAGACT TCTTCACAG TGTCC 2487GYRA_NC000117_514_536_F TCAGGCATTGC 287 GYRA_NC000117_640_660_RTGCTGTAGGGAAA 1251 GGTTGGGATGG TCAGGGCC 2488 GYRA_NC000117_801_827_FTGTGAATAAAT 636 GYRA_NC000117_871_893_R TTGTCAGACTCAT 1419 CACGATTGATTCGCGAACATC GAGCA 2489 GYRA_NC002952_219_242_F TGTCATGGGTA 632GYRA_NC002952_319_345_R TCCATCCATAGAA 1010 AATATCACCCT CCAAAGTTACCTT CAG 2490 GYRA_NC002952_964_983_F TACAAGCACTC 176 GYRA_NC002952_1024_1041_RTCGCAGCGTGCGT 1073 CCAGCTGCA GGCAC 2491 GYRA_NC002952_1505_1520_FTCGCCCGCGAG 366 GYRA_NC002952_1546_1562_R TTGGTGCGCTTGG 1416 GACGT CGTA2492 GYRA_NC002952_59_81_F TCAGCTACATC 279 GYRA_NC002952_124_143_RTGGCGATGCACTG 1279 GACTATGCGAT GCTTGAG G 2493 GYRA_NC002952_216_239_FTGACGTCATCG 452 GYRA_NC002952_313_333_R TCCGAAGTTGCCC 1032 GTAAGTACCACTGGCCGTC CC 2494 GYRA_NC002952_219_242_2_F TGTACTCGGTA 625GYRA_NC002952_308_330_R TAAGTTACCTTGC 873 AGTATCACCCG CCGTCAACCA CA 2495GYRA_NC002952_115_141_F TGAGATGGATT 453 GYRA_NC002952_220_242_RTGCGGGTGATACT 1236 TAAACCTGTTC TACCGAGTAC ACCGC 2496GYRA_NC002952_517_5_39_F TCAGGCATTGC 287 GYRA_NC002952_643_663_RTGCTGTAGGGAAA 1251 GGTTGGGATGG TCAGGGCC C 2497 GYRA_NC002952_273_2_93_FTCGTATGGCTC 380 GYRA_NC002952_338_360_R TGCGGCAGCACTA 1234 AATGGTGGAGTCACCATCCA 2498 GYRA_NC000912_257_278_F TGAGTAAGTT 462GYRA_NC000912_346_370_R TCGAGCCGAAGTT 1067 CCACCCGCACG CCCTGTCCGTC G2504 ARCC_NC003923- TAGTpGATpAG 229 ARCC_NC003923-2725050- TCpTpTpTpCpGT1116 2725050- AACpTpGTAGG 2724595_214_239P_R ATAAAAAGGACpC2724595_135_161P_F CpACpAATpCp pAATpTpGG GT 2505 PTA_NC003923-TCTTGTpTpTp 417 PTA_NC003923-628885- TACpACpCpTGGT 904629355_629355_237_263P_F ATGCpTpGGTA pTpTpCpGTpTpT AAGCAGATGGpTpGATGATpTpT pGTA 2517 CJMLST_ST1_1852_1883_F TTTGCGGATGA 708CJMLST_ST1_1945_1977_R TGTTTTATGTG 1355 AGTAGGTGCCT TAGTTGAGCTTACATCTTTTTGC TTACTACATGAGC 2518 CJMLST_ST1_2963_2992_F TGAAATTGCTA 428CJMLST_ST1_3073_3097_R TCCCCATCTCCGCA 1020 CAGGCCCTTTA AAGACAATAAAGGACAAGG 2519 CJMLST_ST1_2350_2378_F TGCTTTTGATG 535CJMLST_ST1_2447_2481_R TCTACAACACT 1117 GTGATGCAGA TGATTGTAATTTGTCGTTTGG CCTTGTTCTTT 2520 CJMLST_ST1_654_684_F TATGTCCAAGA 240CJMSLT_ST1_725_756_R TCGGAAACAAAGA 1084 AGCATAGCAAA ATTCATTTTCTGGAAAAGCAAT TCCAAA 2521 CJMSLT_ST1_360_395_F TCCTGTTATTC 347CJMLST_ST1_454_457_R TGCTATATGCTAC 1245 CTGAAGTAGTT AACTGGTTCAAAAAATCAAGTTTG ACATTAAG TTA 2522 CJMSLT_ST1_1231_1258_F TGGCAGTTTTA 564CJMSLT_ST1_1312_1340_R TTTAGCTACTATT 1427 CAAGGTGCTGT CTAGCTGCCATTTTTCATC CCA 2523 CJMSLT_ST1_3543_3574_F TGCTGTAGCTT 529CJMLST_ST1_3656_3685_R TCAAAGAACCAGC 1427 ATCGCGAAATG ACCTAATTCATCATCTTTGATTT TTTA 2524 CJMLST_ST1_1_17_F TAAAACTTTTG 145CJMSLT_ST1_55_84_R TGTTCCAATAGCA 1348 CCGTAATGATG GTTCCGCCCAAATGGTGAAGATAT TGAT 2525 CJMSLT_ST1_1312_1342_F TGGAAATGGCA 538CJMSLT_ST1_1383_1417_R TTTCCCCGATC 1432 GCTAGAATAGT TAAATTTGGATAAAGCTAAAAT GCCATAGGAAA 2526 CJMSLT_ST1_2254_2286_F TGGGCCTAATG 582CJMSLT_ST1_2352_2379_R TCCAAACGATC 996 GGCTTAATATC TGCATCACCATCAAATGAAAATTG AAAG 2527 CJMSLT_ST1_1380_1411_F TGCTTTCCTAT 534CJMSLT_ST1_1486_1520_R TGCATGAAGCATA 1205 GGCTTATCCAA AAAACTGTATCAAATTTAGATCG GTGCTTTTA 2528 CJMLST_ST1_3413_3437_F TTGTAAATGCC 692CJMLST_ST1_3511_3542_R TGCTTGCTCAAAT 1257 GGTGCTTCAGA CATCATAAACAAT TCCTAAAGC 2529 CJMSLT_ST1_1130_1156_F TACGCGTCTTG 189 CJMSLT_ST1_1203_RTAGGATGAGCATT 920 AAGCGTTTCGTTA ATCAGGGAAAGAA TGA AGAATC 2530CJMSLT_ST1_2840_2872_F TGGGGCTTTGC 591 CJMSLT_ST1_2940_2973_RTAGCGATTTCT 917 TTTATAGTTTT ACTCCTAGAGTTG TTACATTTAAG AAATTTCAGG 2531CJMSLT_ST1_2058_2084_F TATTCAAGGTG 241 CJMSLT_ST1_2131_2162_RTTGGTTCTTACTT 1417 GTCCTTTGATG GTTTTGCATAAAC CATGT TTTCCA 2532CJMSLT_ST1_553_585_F TCCTGATGCTC 344 CJMLST_ST1_655_685_R TATTGCTTTTTTT942 AAAGTGCTTTT GCTATGCTTCTTG TTAGATCCTTT GACAT 2564 GTLA_NC002163-TCATGTTGAGC 299 GTLA_NC002163-1604930- TTTTGCTCATGAT 1443 1604930-TTAAACCTATA 1604529_352_380_R CTGCATGAAGCAT 1604529_306_338_FGAAGTAAAAGC AAA 2565 UNCA_NC002163- TCCCCCACGCT 322UNCA_NC002163-112166- TCGACCTGGAGGA 1065 112166- TTAATTGTTTT112647_146_171_R CGACGTAAAATCA 112647_80_113_F ATGATGATTTG CGACGTAAAATCAAG 2566 UNCA_NC002163- TAATGATGAAT 170 UNCA_NC002316-112166- TGGGATAACAT1285 112166- TAGGTGCGGGT 112647_294_329_R TGGTTGGAATATA 112647_233_305_FTCTTT AGCAGAAACATC 2567 PGM_NC002163- TCTTGATACTT 414PGM_NC002163-327773- TCCATCGCCAGTT 1012 327773- GTAATGTGGGC328270_365_396_R TTTGCATAATCGC 328270_273_305_F GATAAATATGT TAAAAA 2568TKT_NC002163- TTATGAAGCGT 661 TKT_NC002163-1569415- TCAAAACGCATTT 9461569415- GTTCTTTAGCA 1569873_350_383_R TTACATCTTCGTT 1569873_255_284_FGGACTTCA AAAGGCTA 2570 GTLA_NC002163- TCGTCTTTTTG 381GLTA_NC002163-1604930- TGTTCATGTTTAA 1347 1604930- ATTCTTTCCCT1604529_109_142_R ATGATCAGGATAA 1604529_39_68_F GATAATGC AAAGCACT 2571TKT_NC002163- TGATCTTAAAA 472 TKT_NC002163-1569415- TGCCATAGCAAAG 12141569415- ATTTCCGCCAA 1569903_139_162_R CCTACAGCATT 1569903_33_62_FCTTCATTC CCTACAGCATT 2572 TKT_NC002163- TAAGGTTTATT 164TKT_NC002163-1569415- TACATCTCCTTCG 886 1569415- GTCTTTGTGGA1569903_313_345_R ATAGAAATTTCAT 1569903_207_239_F GATGGGGATTT TGCTATC2573 TKT_NC002163- TAGCCTTTAAC 213 TKT_NC002163-1569415- TAAGACAAGGTTT865 1569415- GAAAATGTAAA 1569903_449_481_R TGTGGATTTTTTA1569903_350_383_F AATGCGTTTTG GCTTGTT A 2574 TKT_NC002163- TTCAAAAACTC665 TKT_NC002163-1569415- TTGCCATAGCAAA 1405 1569415- CAGGCCATCCT1569903_139_163_R GCCTACAGCATT 1569903_60_92_F GAAATTTCAAC 2575GTLA_NC002163- TCGTCTTTTTG 382 GLTA_NC002163-1604930- TGCCATTTCCATG 12161604930- ATTCTTTCCCT 1604529_139_168_R TACTCTTCTCTAA 1604529_39_70_FGATAATGCTC CATT 2576 GLYA_NC002163- TCAGCTATTTT 281GLYA_NC002163-367572- ATTGCTTCTTACT 756 367572- TCCAGGTATCC368079_476_508_R TGCTTAGCATAAA 368079_386_414_F AAGGTGG TTTTCCA 2577GLYA_NC002163- TGGTGCGAGTG 611 GLYA_NC002163-367572- TGCTCACCTGCTA 1246367572- CTTATGCTCGT 368079_242_270_R CAACAAGTCCAGC 368079_148_174_FATTAT AAT 2578 GLYA_NC002163- TGTAAGCTCTA 622 GLYA_NC002163-367572-TTCCACCTTGGAT 1381 367572- CAACCCACAAA 368079_384_416_R ACCTGGAAAAATA368079_298_327_F ACCTTACG GCTGAAT 2579 GLYA_NC002163- TGGTGGACATT 614GLYA_NC002163-367572- TCAAGCTCTACAC 961 367572- TAACACATGGT368079_52_81_R CATAAAAAAAGCT 368079_1_27_F GCAAA CTCA 2580 PGM_NC002163-TGAGCAATGGG 455 PGM_NC002163-327746- TTTGCTCTCCGCC 1438 327746-GCTTTGAAAGA 328270_356_379_R AAAGTTTCCAC 328270_254_285_F AAGAATTTTTAAAT 2581 PGM_NC002163- TGAAAAGGGTG 425 PGM_NC002163-327746-TGCCCCATTGCTC 1219 327746- AAGTAGCAAAT 328270_241_267_R TCATGATAGTAGT328270_153_182_F GGAGATAG AGCTAC 2582 PGM_NC002163- TGGCCTAATGG 568PGM_NC002163-327746- TGCACGCAAACGC 1200 327746- GCTTAATATCA328270_79_102_R TTTACTTCAGC 328270_19_50_F ATGAAAATTG 2583UNCA_NC002163- TAAGCATGCTG 160 UNCA_NC002163-112166- TGCCCTTTCTAAA 1220112166- TGGCTTATCGT 112647_196_225_R AGTCTTGAGTGAA 112647_114_141_FGAAATG GATA 2584 UNCA_NC002163- TGCTTCGGATC 532 UBCA_NC002163-112166-TGCATGCTTACTC 1206 112166- CAGCAGCACTT 532 112647_88_123_R AAATCATCATAAA112647_3_29_F CAATA CAATTAAAGC 2585 ASPA_NC002163- TTAATTTGCCA 652ASPA_NC002163-96692- TGCAAAAGTAACG 1192 96692- AAAATGCAACC97166_403_432_R GTTACATCTGCTC 97166_308_335_F AGGTAG CAAT 2586ASPA_NC002163- TCGCGTTGCAA 370 ASPA_NC002163-966692- TCATGATAGAACT 99196692- CAAAACTTTCT 97166_316_346_R ACCTGGTTGCATT 97166_228_258_FAAAGTATGT TTTGG 2587 GLNA_NC002163- TGGAATGATGA 547GLNA_NC002163-658085- TGAGTTTGAACCA 1176 658085- TAAAGATTTCG657609_340_371_R TTTCAGAGAGCGA 657609_244_275_F CAGATAGCTA ATATCTAC 2588TKT_NC002163- TCGCTACAGGC 371 TKT_NC002163-1569415- TCCCCATCTCCGC 10201569415- CCTTTAGGACA 1569903_212_236_R AAAGACAATAAA 1569903_107_130_FCAGATAGCTA ATATCTAC 2589 TKT_NC002163- TGTTCTTTAGC 642TKT_NC002163-1569415- TCCTTGTGCTTCA 1057 1569415- AGGACTTCACA1569903_361_393_R AAACGCATTTTTA 1569903_265_296_F AACTTGATAA CATTTTC2590 GLYA_NC002163- TGCCTATCTTT 505 GLYA_NC002163-367572- TCCTCTTGGGCCA1047 367572- TTGCTGATATA 368095_317_340_R CGCAAAGTTTT 368095_214_246_FGCACATATTGC CGCAAAGTTTT 2591 GLYA_NC002163- TCCTTTGATGC 353GLYA_NC002163-367572- TCTTGAGCATTGG 1141 367572- ATGTAATTGCT368095_485_516_R TTCTTACTTGTTT 368095_415_444_F GCAAAAGC TGCATA 2592PGM_NC002163_21_54_F TCCTAATGGAC 332 PGM_NC002163_116_142_RTCAAACGATCCGC 949 TTAATATCAAT ATCACCATCAAAA GAAAATTGTGG G G 2593PGM_NC002163_149_176_F TAGATGAAAAA 207 PGM_NC002163_247_277_RTCCCCTTTAAAGC 1023 GGCGAAGTGGC ACCATTACTCATT TAATGG ATAGT 2594GLNA_NC002163- TGTCCAAGAAG 633 GLNA_NC002163-658085- TCAAAAACAAAGA658085- CATAGCAAAAA 657609_148_179_R ATTCATTTTCTGG 657609_79_106_FAAGCAA TCCAAA 2595 ASPA_NC2163- TCCTGTTATTC 347 ASPA_NC002163-96685-TCAAGCTATATGC 960 96685- CTGAAGTAGTT 97196_467_497_R TACAACTGGTTCA97196_367_402_F AATCAAGTTTG AAAAC TTA 2596 ASPA_NC002163- TGCCGTAATGA502 ASPA_NC002163-96685- TACAACCTTCGGA 880 96685- TAGGTGAAGAT97196_95_127_R TAATCAGGATGAG 97196_1_33_F ATACAAAGAGT AATTAAT 2597ASPA_NC002163- TGGAACAGGAA 540 ASPA_NC002163-96685- TAAGCTCCCGTAT 87296685- TTAATTCTCAT 97196_185_210_R CTTGAGTCGCCTC 97196_85_117_FCCTGATTATCC 2598 PGM_NC002163- TGGCAGCTAGA 563 PGM_NC002163-327746-TCACGATCTAAAT 975 327746- ATAGTAGCTAA 328270_230_261_R TTGGATAAGCCAT328270_165_195_F AATCCCTAC AGGAAA 2599 PGM_NC002163- TGGGTCGTGGT 593PGM_NC002163-327746- TTTTGCTCATGAT 1443 327746- TTTACAGAAAA328270_353_381_R CTGCATGAAGCAT 328270_252_286_F TTTCTTATATA AAA 2600PGM_NC002163- TGGGATGAAAA 577 PGM_NC002163-327746- TGATAAAAAGCAC 1178327746- AGCGTTCTTTT 328270_95_123_R TAAGCGATGAAAC 328270_1_30_F ATCCATGAAGC 2601 PGM_NC002163- TAAACACGGCT 146 PGM_NC002163-327746-TCAAGTGCTTTTA 963 327746- TTCCTATGGCT 328270_314_345_R CTTCTATAGGTTT328270_220_250_F TATCCAAAT AAGCTC 2602 UNCA_NC002163- TGTAGCTTATC 628UNCA_NC002163-112166- TGCTTGCTCTTTC 1258 112166- GCGAAATGTCT112647_199_229_R AAGCAGTCTTGAA 112647_123_152_F TTGATTTT TGAAG 2603UBCA_NC002163- TCCAGATGGAC 313 UNCA_NC002163-112166- TCCGAAACTTGTT 1031112166- AAATTTTCTTA 112647_430_461_R TGTAGCTTTAATT 112647_333_365_FGAAACTGATTT TGAGC 2734 GYRA_AY291534_237_264_F TCACCCTCATG 265GYRA_AY291534_268_288_R TTGCGCCATACGT 1407 GTGATTCAGCT ACCATCGT GTTTAT2735 GYRA_AY291534_224_252_F TAATCGGTAAG 167 GYRA_AY291534_256_285_RTGCCATACGTACC 1213 TATCACCCTCA ATCGTTTCATAAA TGGTGAT CAGC 2736GYRA_AY291534_170_198_F TAGGAATTACG 221 GYRA_AY291534_268_288_RTTGCGCCATACGT 1407 GCTGATAAAGC ACCATCGT GTATAAA 2737GYRA_AY291534_224_252_F TAATCGGTAAG 167 GYRA_AY291534_319_346_RTATCGACAGATCC 935 ATCACCCTCAT AAAGTTACCATGC GGTGAT CC 2738GYRA_NC002953-7005- TAAGGTATGAC 163 GYRA_NC002953-7005- TCTTGAGCCATAC1142 9668_166_195_F ACCGGATAAAT 9668_265_287_R GTACCATTGC CATATAAAGTACCATTGC 2739 GYRA_NC002953-7005- TAATGGGTAAA 171 GYRA_NC002953-7005-TATCCATTGAACC 933 9668_221_249_F TATCACCCTCA 9668_316_343_RAAAGTTACCTTGG TGGTGAC CC 2740 GYRA_NC002953-7005- TAATGGGTAAA 171GYRA_NC002953-7005- TAGCCATACGTAC 912 9668_221_249_F TATCACCCTCA9668_253_283_R CATTGCTTCATAA TGGTGAC AATAGA 2741 GYRA_NC002953-7005-TCACCCTCATG 264 GYRA_NC002953-7005- TCTTGAGCCATAC 1142 9668_234_261_FGTGACTCATCT 9668_265_287_R GTACCATTGC ATTTAT 2842 CAPC_AF188935-TGGGATTATTG 578 CAPC_AF188935-56074- TGGTAACCCTTGT 1299 56074-TTATCCTGTTA 55628_348_378_R CTTTGAATTGTAT 55628_271_304_F TGCCATTTGAGTTGCA A 2843 CAPC_AF188935- TGATTATTGTT 476 CAPC_AF188935-56074-TGTAACCCTTGTC 1344 56074- ATCCTGTTATG 55628_349_377P_R TTTGAATpTpGTA55628_273_303P_F CpCpATpTpTp TpTpTpGC GAG 2844 CAPC_AF188935-TCCGTTGATTA 331 CAPC_AF188935-56074- TGTTAATGGTAAC 1344 56074-TTGTTATCCTG 55628_349_384_R CCTTGTCTTTGAA 55628_268_303_F TTATGCCATTTTTGTATTTGC GAG 2845 CAPC_AF188935- TCCGTTGATTA 331 CAPC_AF188935-56074-TAACCCTTGTCTT 860 56074- TTGTTATCCTG 55628_337_375_R TGAATTGTATTTG55628_268_303_F TTATGCCATTT CAATTAATCCTGG GAG 2846 PARC_X95819_33_58_FTCAAAAAAAT 302 PARC_X95819_121_153_R TAAAGGATAGCGG 852 CAGCGCGTACATAACTAAATGGCT GTGG GAGCCAT 2847 PARC_X95819_65_92_F TACTTGGTAAA 199PARC_X95819_157_178_R TACCCCAGTTCCC 889 TACCACCCACA CTGACCTTC TGGTGA2848 PARC_X95819_69_93_F TGGTAAATACC 596 PARC_X95819_97_128_RTGAGCCATGAGTA 1169 ACCCACACATG CCATGGCTTCATA GTGAC ACATGC 2849PARC_NC003997- TTCCGTAAGTC 668 PARC_NC003997-3362578- TCCAAGTTTGACT 10013362578- GGCTAAAACAG 3365001_256_283_R TAAACGTACCATC 3365001_181_205_FTCG GC 2850 PARC_NC003997- TGTAACTATCA 621 PARC_NC003997-3362578-TCGTCAACACTAC 1099 3362578- CCCGCACGGTG 3365001_304_335_R CATTATTACCATG3365001_217_240_F AT CATCTC 2851 PARC_NC003997- TGTAACTATCA 621PARC_NC003997-3362578- TGACTTAAACGTA 1162 3362578- CCCGCACGGTG3365001_244_275_R CCATCGCTTCATA 3365001_217_240_F AT TCATCTC 2852GYRA_AY642140_1_24_F TAAATCTGCCC 150 GYRA_AY642140_71_100_RTGCTAAAGTCTTG 1242 CGTGTCGTTGG AGCCATACGAACA TGAC ATGG 2853GYRA_AY642140_26_54_F TAATCGGTAAA 166 GYRA_AY642140_121_146_RTCGATCGAACCGA 1069 TATCACCCGCA AGTTACCCTGACC TGGTGAC 2854GYRA_AY642140_26_54_F TAATCGGTAAA 166 GYRA_AY642140_58_89_RTGAGCCATACGAA 1168 TATCACCCGCA CAATGGTTTCATA TGGTGAC AACAGC 2860CYA_AF065404_1348_1379_F TCCAACGAAGT 305 CYA_AF065404_1448_1472_RTCAGCTGTTAACG 983 ACAATACAAGA GCTTCAAGACCC CAAAAGAAGG 2861FEF_BA_AF065404_751_781_F TCGAAAGCTTT 354 LEF_BA_AF065404_843_881_RTCTTTAAGTTCTT 1144 TGCATATTATA CCAAGGATAGATT TCGAGCCAC TATTTCTTGTTCG2862 LEF_BA_AF065404_762_788_F TGCATATTATA 498 LEF_BA_AF065404_843_881_RTCTTTAAGTTCTT 1144 TCGAGCCACAG CCAAGGATAGATT CATCG TATTTCTTGTTCG 2917MUTS_AY698802_106_125_F TCCGCTGAATC 326 MUTS_AY698802_172_193_RTGCGGTCTGGCGC 1237 TGTCGCCGC ATATAGGTA 2918 MUTS_AY698802_172_192_FTACCTATATGC 187 MUTS_AY698802_228_252_R TCAATCTCGACTT 965 GCCAGACCGCTTTGTGCCGGTA 2919 MUTS_AY698802_228_252_F TACCGGCGCAA 186MUTS_AY698802_314_342_R TCGGTTTCAGTCA 1097 AAAGTCGAGAT TCTCCACCATAAA TGGGGT 2920 MUTS_AY698802_315_342_F TCTTTATGGTG 419 MUTS_AY698802_413_433_RTGCCAGCGACAGA 1210 GAGATGACTGAAA CCATCGTA CCGA 2921MUTS_AY698802_394_411_F TGGGCGTGGAA 585 MUTS_AY698802_497_519_RTCCGGTAACTGGG 1040 CGTCCAC TCAGCTCGAA 2922 AB_MLST-11- TGGGcGATGCT 583AB_MLST-11- TAGTATCACCACG 923 OIF007_991_1018_F GCgAAATGGTTOIF007_1110_1137_R TACACCCGGATCA AAAAGA GT 2927 GAPA_NC002505_694_721_FTCAATGAACGA 259 GAPA_NC_002505_29_58_R_1 TCCTTTATGCAAC 1060 CCAACAAGTGATTGGTATCAACAG TTGATG GAAT 2928 GAPA_NC002505_694_721_2_F TCGATGAACGA 361GAPA_NC002505_769_798_2_R TCCTTTATGCAAC 1061 CCAACAAGTGA TTGGTATCAACCGTTGATG GAAT 2929 GAPA_NC002505_694_721_2_F TCGATGAACGA 361GAPA_NC002505_769_798_3_R TCCTTTATGCAAC 1059 CCAACAAGTGA TTAGTATCAACCGTTGATG GAAT 2932 INFB_EC_1364_1394_F TTGCTCGTGGT 688 INFB_EC_1439_1468_RTTGCTGCTTTCGC 1410 GCACAAGTAA ATGGTTAATCGCT CGGATATTAC TCAA 2933INFB_EC_1364_1394_2_F TTGCTCGTGGT 689 INFB_EC_1439_1468_R TTGCTGCTTTCGC1410 GCAIAAGTAA ATGGTTAATCGCT CGGATATIAC TCAA 2934 INFB_EC_80_110_FTTGCCCGCGGT 685 INFB_EC_1439_1468_R TTGCTGCTTTCGC 1410 GCGGAAGTAACATGGTTAATCGCT CGATATTAC TCAA 2949 ACS_NC002516- TCGGCGCCTGC 376ACS_NC002516-970624- TGGACCACGCCGA 1265 970624- CTGATGA 971013_364_383_RAGAACGG 2950 ARO_NC002516_26883- TCACCGTGCCG 267 ARO_NC002516-26883-TGTGTTGTCGCCG 1341 27380_4_26_F TTCAAGGAAGA 27380_111_128_R CGCAG G 2951ARO_NC002516-26883- TTTCGAAGGGC 705 ARO_NC002516-26883- TCCTTGGCATACA1056 27380_356_377_F CTTTCGACCTG 27380_459_484_R TCATGTCGTAGCA 2952GUA_NC002516- TGGACTCCTCG 551 GUA_NC002516-4226546- TCGGCGAACATGG 10914226546- GTGGTCGC 4226174_127_146_R CCATCAC 2953 GUA_NC002516-TGACCAGGTGA 448 GUA_NC002516-4226546- TGCTTCTCTTCCG 1256 4226546-TGGCCATGTTC 4226174_214_233_R GGTCGGC 4226174_120_142_F G 2954GUA_NC002516- TTTTGAAGGTG 710 GUA_NC002516-4226546- TGCTTGGTGGCTT 12594226546- ATCCGTGCCAA 4226174_265_287_R CTTCGTCGAA 4226174_155_178_F CG2955 GUA_NC002516- TTCCTCGGCCG 670 GUA_NC002516-4226546- TGCGAGGAACTTC1229 4226546- CCTGGC 4226174_288_309_R ACGTCCTGC 4226174_190_206_F 2956GUA_NC002516- TCGGCCGCACC 374 GUA_NC002516-4226546- TCGTGGGCCTTGC 11114226546- TTCATCGAAGT 4226174_355_371_R CGGT 2957 MUT_NC002516-TGGAAGTCATC 545 MUT_NC002516-5551158- TCACGGGCCAGCT 978 5551158-AAGCGCCTGGC 5550717_99_116_R CGTCT 5550717_5_26_F AAGCGCCTGGC 2958MUT_NC002516- TCGAGCAGGC 358 MUT_NC002516-5551158- TCACCATGCGCCC 9715551158- GCTGCCG 5550717_256_277_R GTTCACATA 5550717_5_26_F 2959NUO_NC002516- TCAACCTCGGC 249 NUO_NC002516-2984589- TCGGTGGTGGTAG 10952984589- CCGAACCA 2984954_97_117_R CCGATCTC 2960 NUO_NC002516-TACTCTCGGTG 195 NUO_NC002516-2984589- TTCAGGTACAGCA 1376 2984589-GAGAAGCTCGC 2984954_301_326_R GGTGGTTCAGGAT 2961 PPS_NC002516-TCCACGGTCAT 311 PPS_NC002516-1915014- TCCATTTCCGACA 1014 1915014-GGAGCGCTA 1915383_140_165_R CGTCGTTGATCAC 1915383_44_63_P 3962PPS_NC002516- TCGCCATCGTC 365 PPS_NC002516-1915014- TCCTGGCCATCCT 10521915014- ACCAACCG 1915383_341_360_R GCAGGAT 1915383_240_258_F 2963TRP_NC002516- TGCTGGTACGG 527 TRP_NC002516-671831- TCGATCTCCTTG 1071671831- GTCGAGGA 672273_131_150_R GCGTCCGA 672273_24_42_F 2964TRP_NC002516- TGCACATCGTG 490 TRP_NC002516-671831- TGATCTCCATGGC 1182671831- TCCAACGTCAC 672273_362_383_R GCGGATCTT 672273_261_282_F 2972AB_MLST-11- TGGGIGATGCT 592 AB_MLST-11- TAGTATCACCACG 924OIF007_1007_1034_F GCIAAATGGTT OIF007_1126_1153_R TACICCIGGATCA AAAAGAGT 2993 OMPU_NC002505- TTCCCACCGAT 667 OMPU_NC002505_544_567_RTCGGTCAGCAAAA 1094 674828- ATCATGGCTTA CGGTAGCTTGC 675880_428_455_F 2994GAPA_NC002505- TCCTCAATGAA 335 GAPA_NC002505-506780- TTTTCCCTTTATG 1442506780 CGAICAACAAG 507937_769_802_R CAACTTAGTATCA 507937_691_721_FTGATTGATG ACIGGAAT 2995 GAPA_NC002505- TCCTCIATGAA 339GAPA_NC002505-506780- TCCATACCTTTAT 1008 506780- ACGAICAACAA507937_769_803_R GCAACTTIGTAT 507937_691_721_2_F GTGATTGATG CAACIGGAAT2996 GAPA_NC002505- TCCTCGATGAA 396 GAPA_NC002505-506780- TCGGAAATATTCT1085 506780- CCGACCAACAA 507937_785_817_R TTCAATACCTT 507937_692_721_FGTGATTGATTG TATGCAACT 2997 GAPA_NC002505- TCCTCGATGAA 337GAPA_NC002505-506780- TCGGAAATATTCT 1085 506780- CGAICAACAAG507937_785_817_R TTCAATACCTTTA 507937_691_721_3_F TIATTGATG TGCAACT 2998GAPA_NC002505- TCCTCAATGAA 336 GAPA_NC002505-506780- TCGGAAATATTCT 1087506780- TGATCAACAAG 507937_784_817_R TTCAATICCTTTI 507937_691_721_4_FTGATTGATG TGCAACTT 2999 GAPA_NC002505- TCCTCIATGAA 340GAPA_NC002505-506780- TCGGAAATATTCT 1086 506780- IGAICAACAAG507937_784_817_2_R TTCAATACCTTTA 507937_691_721_5_F TIATTGATG TGCAACTT3000 GAPA_NC002505- TCCTCGATGAA 338 GAPA_NC002505-506780- TTTCAATACCTTT1430 506780- TGAICAACAAC 507937_769_805_R GCAACTTIGTATC507937_691_721_6_F AAGTIATTGAT AACIGGAAT G 3001 CTXB_NC002505-TCAGCATATGC 275 CTXB_NC002505-1566967- TCCCGGCTAGAGA 1026 1566967-ACATGGAACAC 1567341_139_163_R TTCTGTATACGA 1567341_46_71_F CTCATTCTGTATACGA 3002 CTXB_NC002505- TCAGCATATGC 274 CTXB_NC002505-1566967-TCCGGCTAGAGAT 1038 1566967- ACATGGAACAC 1567341_132_162_R TCTGTATACGAAA1567341_46_70_F CTC ATATC 3003 CTXB_NC002505- TCAGCATATGC 274CTXB_NC002505-1566967- TGCCGTATACGAA 1225 1566967- ACATGGAACAC1567341_118_150_R AATATCTTATCAT 1567341_46_70_F CTC TTAGCGT 3004TUFB_NC002758- TACAGGCCGTG 180 TUFB_NC002758-615038- TCAGCGTAGTCTA 982615038- TTGAACGTGG 61622_778_809_R ATAATTTACGGAA 616222_684_704_F CATTTC3005 TUFB_NC002758- TGCCGTGTTGA 503 TUFB_NC0027858-615038- TGCTTCAGCGTAG1255 615038- ACGTGGTCAAA 616222_783_813_R TCTAATAATTTAC 616222_688_710_FT GGAAC 3006 TUFB_NC0027858- TGTGGTCAAAT 638 TUFB_NC002758-615038-TGCGTAGTCTAAT 1238 615038- CAAAGTTGGTG 616222_778_807_R AATTTACGGAACA616222_700_726_F AAGA TTTC 3007 TUFB_NC002758- TGGTCAAATCA 607TUFB_NC002758-615038- TGCGTAGTCTAAT 1238 615038- AAGTTGGTGAAG616222_778_807_R AATTTACGGAACA 616222_702_726_F AA TTTC 3008TUFB_NC002758- TGAACGTGGTC 431 TUFB_NC002758-615038- TCACCAGCTTCAG 970615038- AAATCAAAGTT 616222_785_818_R CGTAGTCTAATAA 616222_696_726_FGGTGAAGAA TTTACGGA 3009 TUFB_NC002758- TCGTGTTGAAC 386TUFB_NC002758-615038- TCTTCAGCGCGTA 1134 615038- GTGGTCAAATC616222_778_812_R GTCTAATAATTTA 616222_690_716_F AAAGT CGGAACATTTC 3010MECI-R-NC003923- TCACATATCGT 261 MECI-R_NC003923-41798- TGTGATATGGAGG1332 41798-41609_36_59_F GAGCAATGAAC 41609_89_112_R TGTAGAAGGTG TG 3011MECI-R_NC003923- TGGGCGTGAGC 584 MECI-R_NC003923-41798- TGGGATGGAGGTG1287 41798-41609_40_66_F AATGAACTGAT 41609_81_110_R TAGAAGGTGTTAT TATACCATC 3012 MECI-R_NC003923- TGGACACATAT 549 MECI-R_NC003923-41798-TGGGATGGAGGTG 1286 41798- CGTGAGCAATG 41609_81_110_R TAGAAAGGTGTTA41609_33_60_2_F AACTGA TCATC 3013 MECI-R_NC003923- TGGGTTTACAC 595MECI-R_NC003923-41798- TGGGGATATGGAG 1290 41798-41609_29_60_FATATCGTGAGC 41609_81_113_R TGTAGAAGGTGTT AATGAACTGA ATCATC 3014MUPR_X75439_2409_2513_F TGGGCTCTTTC 587 MUPR_X75439_2548_2570_RTCTGGCTGCGGAA 1130 TCGCTTAAACA GTGAAATCGT CCT 3015MUPR_X75439_2482_2510_F TGGGCTCTTTC 586 MUPR_X75439_2547_2568_RTGGCTGCGGAAGT 1281 TCGCTTAAACA GAAATCGTA CC 3016 MUPR_X75439_2482_2510_FTAGATAATTGG 205 MUPR_X75439_2551_2573_R TAATCTGGCTGCG 876 GCTCTTTCTCGGAAGTGAAAT CTTAAAC 3017 MUPR_X75439_2490_2514_F TGGGCTCTTTC 587MUPR_X75439_2549_2573_R TAATCTGGCTGCG 877 TCGCTTAAACA GAAGTGAAATCG CCT3018 MUPR_X75439_2482_2510_F TAGATAATTGG 205 MUPR_X75439_2559_2589_RTGGTATATTCGTT 1303 GCTCTTTCTCG AATTAATCTGGCT CTTAAAC GCGGA 3019MUPR_X75439_2490_2514_F TGGGCTCTTTC 587 MUPR_X75439_2554_2581_RTCGTTAATTAATC 1112 GCTTAAACACC TGGCTGCGGAAGT T GA 3020 AROE_NC003923-TGATGGCAAGT 474 AROE_NC003923-1674726- TAAGCAATACCTT 1378 1674726-GGATAGGGTAT 1674277_309_335_R TACTTGCACCACC 1674277_204_232_F AATACAGACCT 3021 AROE_NC003923- TGGCGAGTGGA 570 AROE_NC003923-1674726-TTCATAAGCAATA 1378 1674726- TAGGGTATAAT 1674277_311_339_R CCCTTTACTTGCA1674277_207_232_F ACAG CCAC 3022 AROE_NC003923- TGGCpAAGTpG 572AROE_NC003923-1674726- TAAGCAATACCpT 867 1674726- GATpAGGGTpA1674277_311_335P_R pTpTpACTpTpGC 1674277_207_232P_F TpAATpACpAG pACpCpAC3023 ARCC_NC003923- TCTGAAATGAA 398 ARCC_NC003923-2725050- TCTTCTTCTTTCG1137 2725050- TAGTGATAGAA 2724595_214_245_R TATAAAAAGGACC2724595_124_155_F CTGTAGGCAC AATTGG 3024 ARCC_NC003923- TGAATAGTGAT 437ARCC_NC003923-2725050- TCTTCTTTTCGTAT 1139 2725050- AGAACTGTAGG2724595_212_242_R AAAAAGGACCAAT 2724595_131_161_F CACAATCGT TGGTT 3025ARCC_NC003923- TGAATAGTGAT 437 ARCC_NC003923-2725050- TGCGCTAATTCTT 12322725050- AGAACTGTAGG 2724595_232_260_R CAACTTCTTCTTT 2724595_131_161_FCACAATCGT CGT 3026 PTA_NC003923- TACAATGCTTG 177 PTA_NC003923-628885-TGTTCTTGATACA 1350 628885- TTTATGCTGGTA 629355_322_351_R CCTGGTTTCGTTT629355_231_259_F AAGCAG TGAT 3027 PTA_NC003923- TACAATGCTTG 177PTA_NC003923-628885- TGGTACACCTGGT 1301 62885- TTTATGCTGGT629355_314_345_R TTCGTTTTGATGA 629355_231_259_F AAAGCAG TTTGTA 3028PTA_NC003923- TCTTGTTTATG 418 PTA_NC003923-628885- TGTTCTTGATACA 1350628885- CTGGTAAAAGC 629355_322_351_R CCTGGTTTCGTTT 629355_237_263_FAGATGG TGAT

Primer pair name codes and reference sequences are shown in Table 3. Theprimer name code typically represents the gene to which the given primerpair is targeted. The primer pair name may include specific coordinateswith respect to a reference sequence defined by an extraction of asection of sequence or defined by a GenBank gi number, or thecorresponding complementary sequence of the extraction, or the entireGenBank gi number as indicated by the label “no extraction.” Where “noextraction” is indicated for a reference sequence, the coordinates of aprimer pair named to the reference sequence are with respect to theGenBank gi listing. Gene abbreviations are shown in bold type in the“Gene Name” column.

To determine the exact primer hybridization coordinates of a given pairof primers on a given bioagent nucleic acid sequence and to determinethe sequences, molecular masses and base compositions of anamplification product to be obtained upon amplification of nucleic acidof a known bioagent with known sequence information in the region ofinterest with a given pair of primers, one with ordinary skill inbioinformatics is capable of obtaining alignments of the primers of thepresent invention with the GenBank gi number of the relevant nucleicacid sequence of the known bioagent. For example, the reference sequenceGenBank gi numbers (Table 3) provide the identities of the sequenceswhich can be obtained from GenBank. Alignments can be done using abioinformatics tool such as BLASTn provided to the public by NCBI(Bethesda, Md.). Alternatively, a relevant GenBank sequence may bedownloaded and imported into custom programmed or commercially availablebioinformatics programs wherein the alignment can be carried out todetermine the primer hybridization coordinates and the sequences,molecular masses and base compositions of the amplification product. Forexample, to obtain the hybridization coordinates of primer pair number2095 (SEQ ID NOs: 456:1261), First the forward primer (SEQ ID NO: 456)is subjected to a BLASTn search on the publicly available NCBI BLASTwebsite. “RefSeq_Genomic” is chosen as the BLAST database since the ginumbers refer to genomic sequences. The BLAST query is then performed.Among the top results returned is a match to GenBank gi number 21281729(Accession Number NC_(—)003923). The result shown below, indicates thatthe forward primer hybridizes to positions 1530282.1530307 of thegenomic sequence of Staphylococcus aureus subsp. aureus MW2 (representedby gi number 21281729).

-   Staphylococcus aureus subsp. aureus MW2, complete genome    Length=2820462-   Features in this part of subject sequence:    -   Panton-Valentine leukocidin chain F precursor-   Score=52.0 bits (26), Expect=2e-05-   Identities=26/26 (100%), Gaps=0/26 (0%)-   Strand=Plus/Plus

The hybridization coordinates of the reverse primer (SEQ ID NO: 1261)can be determined in a similar manner and thus, the bioagent identifyingamplicon can be defined in terms of genomic coordinates. Thequery/subject arrangement of the result would be presented inStrand=Plus/Minus format because the reverse strand hybridizes to thereverse complement of the genomic sequence. The preceding sequenceanalyses are well known to one with ordinary skill in bioinformatics andthus, Table 3 contains sufficient information to determine the primerhybridization coordinates of any of the primers of Table 2 to theapplicable reference sequences described therein.

TABLE 3 Primer Name Codes and Reference Sequence Reference GenBank giPrimer name code Gene Name Organism number 16S_EC 16S rRNA (16Sribosomal RNA gene) Escherichia coli 16127994 23S_EC 23S rRNA (23Sribosomal RNA gene) Escherichia coli 16127994 CAPC_BA capC (capsulebiosynthesis gene) Bacillus anthracis 6470151 CYA_BA cya (cyclic AMPgene) Bacillus anthracis 4894216 DNAK_EC dnaK (chaperone dnaK gene)Escherichia coli 16127994 GROL_EC groL (chaperonin groL) Escherichiacoli 16127994 HFLB_EC hflb (cell division protein peptidase Escherichiacoli 16127994 ftsH) INFB_EC infB (protein chain initiation factorEscherichia coli 16127994 infB gene) LEF_BA lef (lethal factor) Bacillusanthracis 21392688 PAG_BA pag (protective antigen) Bacillus anthracis21392688 RPLB_EC rplB (50S ribosomal protein L2) Escherichia coli16127994 RPOB_EC rpoB (DNA-directed RNA polymerase beta Escherichia coli6127994 chain) RPOC_EC rpoC (DNA-directed RNA polymerase Escherichiacoli 16127994 beta' chain) SP101ET_SPET_11 Artificial SequenceConcatenation Artificial 15674250 comprising: Sequence* - gki (glucosekinase) partial gene gtr (glutamine transporter protein) sequences ofmurI (glutamate racemase) Streptococcus mutS (DNA mismatch repairprotein) pyogenes xpt (xanthine phosphoribosyl transferase) yqiL(acetyl-CoA-acetyl transferase) tkt (transketolase) SSPE_BA sspE (smallacid-soluble spore Bacillus anthracis 30253828 protein) TUFB_EC tufB(Elongation factor Tu) Escherichia coli 16127994 VALS_EC valS(Valyl-tRNA synthetase) Escherichia coli 16127994 ASPS_EC aspS(Aspartyl-tRNA synthetase) Escherichia coli 16127994 CAF1_AF053947 caf1(capsular protein caf1) Yersinia pestis 2996286 INV_U22457 inv (invasin)Yersinia pestis 1256565 LL_NC003143 Y. pestis specific chromosomalgenes - Yersinia pestis 16120353 difference region BONTA_X52066 BoNT/A(neurotoxin type A) Clostridium 40381 botulinum MECA_Y14051 mecAmethicillin resistance gene Staphylococcus 2791983 aureus TRPE_AY094355trpE (anthranilate synthase (large Acinetobacter 20853695 component))baumanii RECA_AF251469 recA (recombinase A) Acinetobacter 9965210baumanii GYRA_AF100557 gyrA (DNA gyrase subunit A) Acinetobacter 4240540baumanii GYRB_AB008700 gyrB (DNA gyrase subunit B) Acinetobacter 4514436baumanii WAAA_Z96925 waaA (3-deoxy-D-manno-octulosonic-acidAcinetobacter 2765828 transferase) baumanii CJST_CJ Artificial SequenceConcatenation Artificial 15791399 comprising: Sequence* - tkt(transketolase) partial gene glyA (serine hydroxymethyltransferase)sequences of gltA (citrate synthase) Campylobacter aspA (aspartateammonia lyase) jejuni glnA (glutamine synthase) pgm (phosphoglyceratemutase) uncA (ATP synthetase alpha chain) RNASEP_BDP RNase P(ribonuclease P) Bordetella 33591275 pertussis RNASEP_BKM RNase P(ribonuclease P) Burkholderia 53723370 mallei RNASEP_BS RNase P(ribonuclease P) Bacillus subtilis 16077068 RNASEP_CLB RNase P(ribonuclease P) Clostridium 18308982 perfringens RNASEP_EC RNase P(ribonuclease P) Escherichia coli 16127994 RNASEP_RKP RNase P(ribonuclease P) Rickettsia 15603881 prowazekii RNASEP_SA RNase P(ribonuclease P) Staphylococcus 15922990 aureus RNASEP_VBC RNase P(ribonuclease P) Vibrio cholerae 15640032 ICD_CXB icd (isocitratedehydrogenase) Coxiella burnetii 29732244 IS1111A multi-locus IS1111Ainsertion element Acinetobacter 29732244 baumannii OMPA_AY485227 ompA(outer membrane protein A) Rickettsia 40287451 prowazekii OMPB_RKP ompB(outer membrane protein B) Rickettsia 15603881 prowazekii GLTA_RKP gltA(citrate synthase) Vibrio cholerae 15603881 TOXR_VBC toxR (transcriptionregulator toxR) Francisella 15640032 tularensis ASD_FRT asd (Aspartatesemialdehyde Francisella 56707187 dehydrogenase) tularensis GALE_FRTgalE (UDP-glucose 4-epimerase) Shigella flexneri 56707187 IPAH_SGF ipaH(invasion plasmid antigen) Campylobacter 30061571 jejuni HUPB_CJ hupB(DNA-binding protein Hu-beta) Coxiella burnetii 15791399 AB_MLSTArtificial Sequence Concatenation Artificial Sequenced comprising:Sequence* - in-house trpE (anthranilate synthase component partial gene(SEQ ID I)) sequences of NO: 1444) adk (adenylate kinase) AcinetobactermutY (adenine glycosylase) baumannii fumC (fumarate hydratase) efp(elongation factor p) ppa (pyrophosphate phospho- hydratase MUPR_X75439mupR (mupriocin resistance gene) Staphylococcus 438226 aureusPARC_X95819 parC (topoisomerase IV) Acinetobacter 1212748 baumanniiSED_M28521 sed (enterotoxin D) Staphylococcus 1492109 aureusPLA_AF053945 pla (plasminogen activator) Yersinia pestis 2996216SEJ_AF053140 sej (enterotoxin J) Staphylococcus 3372540 aureusGYRA_NC000912 gyrA (DNA gyrase subunit A) Mycoplasma 13507739 pneumoniaeACS_NC002516 acsA (Acetyl CoA Synthase) Pseudomonas 15595198 aeruginosaARO_NC002516 aroE (shikimate 5-dehydrogenase Pseudomonas 15595198aeruginosa GUA_NC002516 guaA (GMP synthase) Pseudomonas 15595198aeruginosa MUT_NC002516 mutL (DNA mismatch repair protein) Pseudomonas15595198 aeruginosa NUO_NC002516 nuoD (NADH dehydrogenase I chain C, D)Pseudomonas 15595198 aeruginosa PPS_NC002516 ppsA (Phosphoenolpyruvatesynthase) Pseudomonas 15595198 aeruginosa TRP_NC002516 trpE(Anthranilate synthetase Pseudomonas 15595198 component I) aeruginosaOMP2_NC000117 ompB (outer membrane protein B) Chlamydia 15604717trachomatis OMPA_NC000117 ompA (outer membrane protein B) Chlamydia15604717 trachomatis GYRA_NC000117 gyrA (DNA gyrase subunit A) Chlamydia15604717 trachomatis CTXA_NC002505 ctxA (Cholera toxin A subunit) Vibriocholerae 15640032 CTXB_NC002505 ctxB (Cholera toxin B subunit) Vibriocholerae 15640032 FUR_NC002505 fur (ferric uptake regulator protein)Vibrio cholerae 15640032 GAPA_NC_002505 gapA (glyceraldehyde-3-phosphateVibrio cholerae 15640032 dehydrogenase) GYRB_NC002505 gyrB (DNA gyrasesubunit B) Vibrio cholerae 15640032 OMPU_NC002505 ompU (outer membraneprotein) Vibrio cholerae 15640032 TCPA_NC002505 tcpA (toxin-coregulatedpilus) Vibrio cholerae 15640032 ASPA_NC002163 aspA (aspartate ammonialyase) Campylobacter 15791399 jejuni GLNA_NC002163 glnA (glutaminesynthetase) Campylobacter 15791399 jejuni GLTA_NC002163 gltA (glutamatesynthase) Campylobacter 15791399 jejuni GLYA_NC002163 glyA (serinehydroxymethyltransferase) Campylobacter 15791399 jejuni PGM_NC002163 pgm(phosphoglyceromutase) Campylobacter 15791399 jejuni TKT_NC002163 tkt(transketolase) Campylobacter 15791399 jejuni UNCA_NC002163 uncA (ATPsynthetase alpha chain) Campylobacter 15791399 jejuni AGR-III_NC003923agr-III (accessory gene regulator-III) Staphylococcus 21281729 aureusARCC_NC003923 arcC (carbamate kinase) Staphylococcus 21281729 aureusAROE_NC003923 aroE (shikimate 5-dehydrogenase Staphylococcus 21281729aureus BSA-A_NC003923 bsa-a (glutathione peroxidase) Staphylococcus21281729 aureus BSA-B_NC003923 bsa-b (epidermin biosynthesis proteinStaphylococcus 21281729 EpiB) aureus GLPF_NC003923 glpF (glyceroltransporter) Staphylococcus 21281729 aureus GMK_NC003923 gmk (guanylatekinase) Staphylococcus 21281729 aureus MECI-R_NC003923 mecR1 (truncatedmethicillin Staphylococcus 21281729 resistance protein) aureusPTA_NC003923 pta (phosphate acetyltransferase) Staphylococcus 21281729aureus PVLUK_NC003923 pvluk (Panton-Valentine leukocidin Staphylococcus21281729 chain F precursor) aureus SA442_NC003923 sa442 geneStaphylococcus 21281729 aureus SEA_NC003923 sea (staphylococcalenterotoxin A Staphylococcus 21281729 precursor) aureus SEC_NC003923sec4 (enterotoxin type C precursor) Staphylococcus 21281729 aureusTPI_NC003923 tpi (triosephosphate isomerase) Staphylococcus 21281729aureus YQI_NC003923 yqi (acetyl-CoA C-acetyltransferase Staphylococcus21281729 homologue) aureus GALE_AF513299 galE (galactose epimerase)Francisella 23506418 tularensis VVHA_NC004460 vVhA (cytotoxin, cytolysinprecursor) Vibrio vulnificus 27366463 TDH_NC004605 tdh (thermostabledirect hemolysin A) Vibrio 28899855 parahaemolyticus AGR-II_NC002745agr-II (accessory gene regulator-II) Staphylococcus 29165615 aureusPARC_NC003997 parC (topoisomerase IV) Bacillus anthracis 30260195GYRA_AY291534 gyrA (DNA gyrase subunit A) Bacillus anthracis 31323274AGR-I_AJ617706 agr-I (accessory gene regulator-I) Staphylococcus46019543 aureus AGR-IV_AJ617711 agr-IV (accessory gene regulator-III)Staphylococcus 46019563 aureus BLAZ_NC002952 blaZ (beta lactamase III)Staphylococcus 49482253 aureus ERMA_NC002952 ermA (rRNAmethyltransferase A) Staphylococcus 49482253 aureus ERMB_Y13600 ermB(rRNA methyltransferase B) Staphylococcus 49482253 aureusSEA-SEE_NC002952 sea (staphylococcal enterotoxin A Staphylococcus49482253 precursor) aureus SEA-SEE_NC002952 sea (staphylococcalenterotoxin A Staphylococcus 49482253 precursor) aureus SEE_NC002952 sea(staphylococcal enterotoxin A Staphylococcus 49482253 precursor) aureusSEH_NC002953 seh (staphylococcal enterotoxin H) Staphylococcus 49484912aureus ERMC_NC005908 ermC (rRNA methyltransferase C) Staphylococcus49489772 aureus MUTS_AY698802 mutS (DNA mismatch repair protein)Shigella boydii 52698233 NUC_NC002758 nuc (staphylococcal nuclease)Staphylococcus 57634611 aureus SEB_NC002758 seb (enterotoxin type Bprecursor) Staphylococcus 57634611 aureus SEG_NC002758 seg(staphylococcal enterotoxin G) Staphylococcus 57634611 aureusSEI_NC002758 sei (staphylococcal enterotoxin I) Staphylococcus 57634611aureus TSST_NC002758 tsst (toxic shock syndrome toxin-1) Staphylococcus57634611 aureus TUFB_NC002758 tufB (Elongation factor Tu) Staphylococcus57634611 aureus Note: artificial reference sequences representconcatenations of partial gene extractions from the indicated referencegi number. Partial sequences were used to create the concatenatedsequence because complete gene sequences were not necessary for primerdesign.

Example 2 Sample Preparation and PCR

Genomic DNA was prepared from samples using the DNeasy Tissue Kit(Qiagen, Valencia, Calif.) according to the manufacturer's protocols.

All PCR reactions were assembled in 50 μL reaction volumes in a 96-wellmicrotiter plate format using a Packard MPII liquid handling roboticplatform and M. J. Dyad thermocyclers (MJ research, Waltham, Mass.) orEppendorf Mastercycler thermocyclers (Eppendorf, Westbury, NY). The PCRreaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II(Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl₂, 0.4 M betaine,800 μM dNTP mixture and 250 nM of each primer. The following typical PCRconditions were used: 95° C. for 10 min followed by 8 cycles of 95° C.for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the48° C. annealing temperature increasing 0.9° C. with each of the eightcycles. The PCR was then continued for 37 additional cycles of 95° C for15 seconds, 56° C. for 20 seconds, and 72° C. 20 seconds.

Example 3 Purification of PCR Products for Mass Spectrometry with IonExchange Resin-Magnetic Beads

For solution capture of nucleic acids with ion exchange resin linked tomagnetic beads, 25 μl of a 2.5 mg/mL suspension of BioClone amineterminated superparamagnetic beads were added to 25 to 50 μl of a PCR(or RT-PCR) reaction containing approximately 10 μM of a typical PCRamplification product. The above suspension was mixed for approximately5 minutes by vortexing or pipetting, after which the liquid was removedafter using a magnetic separator. The beads containing bound PCRamplification product were then washed three times with 50 mM ammoniumbicarbonate/50% MeOH or 100 mM ammonium bicarbonate/50% MeOH, followedby three more washes with 50% MEOH. The bound PCR amplicon was elutedwith a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH whichincluded peptide calibration standards.

Example 4 Mass Spectrometry and Base Composition Analysis

The ESI-FTICR mass spectrometer is based on a Bruker Daltonics(Billerica, MA) Apex II 70e electrospray ionization Fourier transformion cyclotron resonance mass spectrometer that employs an activelyshielded 7 Tesla superconducting magnet. The active shielding constrainsthe majority of the fringing magnetic field from the superconductingmagnet to a relatively small volume. Thus, components that might beadversely affected by stray magnetic fields, such as CRT monitors,robotic components, and other electronics, can operate in closeproximity to the FTICR spectrometer. All aspects of pulse sequencecontrol and data acquisition were performed on a 600 MHz Pentium II datastation running Bruker's Xmass software under Windows NT 4.0 operatingsystem. Sample aliquots, typically 15 μl, were extracted directly from96-well microtiter plates using a CTC HTS PAL autosampler (LEAPTechnologies, Carrboro, N.C.) triggered by the FTICR data station.Samples were injected directly into a 10 μl sample loop integrated witha fluidics handling system that supplies the 100 μl/hr flow rate to theESI source. Ions were formed via electrospray ionization in a modifiedAnalytica (Branford, Conn.) source employing an off axis, groundedelectrospray probe positioned approximately 1.5 cm from the metalizedterminus of a glass desolvation capillary. The atmospheric pressure endof the glass capillary was biased at 6000 V relative to the ESI needleduring data acquisition. A counter-current flow of dry N₂ was employedto assist in the desolvation process. Ions were accumulated in anexternal ion reservoir comprised of an rf-only hexapole, a skimmer cone,and an auxiliary gate electrode, prior to injection into the trapped ioncell where they were mass analyzed. Ionization duty cycles greater than99% were achieved by simultaneously accumulating ions in the externalion reservoir during ion detection. Each detection event consisted of 1M data points digitized over 2.3 s. To improve the signal-to-noise ratio(S/N), 32 scans were co-added for a total data acquisition time of 74 s.

The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOF™.Ions from the ESI source undergo orthogonal ion extraction and arefocused in a reflectron prior to detection. The TOF and FTICR areequipped with the same automated sample handling and fluidics describedabove. Ions are formed in the standard MicroTOF™ ESI source that isequipped with the same off-axis sprayer and glass capillary as the FTICRESI source. Consequently, source conditions were the same as thosedescribed above. External ion accumulation was also employed to improveionization duty cycle during data acquisition. Each detection event onthe TOF was comprised of 75,000 data points digitized over 75 μs.

The sample delivery scheme allows sample aliquots to be rapidly injectedinto the electrospray source at high flow rate and subsequently beelectrosprayed at a much lower flow rate for improved ESI sensitivity.Prior to injecting a sample, a bolus of buffer was injected at a highflow rate to rinse the transfer line and spray needle to avoid samplecontamination/carryover. Following the rinse step, the autosamplerinjected the next sample and the flow rate was switched to low flow.Following a brief equilibration delay, data acquisition commenced. Asspectra were co-added, the autosampler continued rinsing the syringe andpicking up buffer to rinse the injector and sample transfer line. Ingeneral, two syringe rinses and one injector rinse were required tominimize sample carryover. During a routine screening protocol a newsample mixture was injected every 106 seconds. More recently a fast washstation for the syringe needle has been implemented which, when combinedwith shorter acquisition times, facilitates the acquisition of massspectra at a rate of just under one spectrum/minute.

Raw mass spectra were post-calibrated with an internal mass standard anddeconvoluted to monoisotopic molecular masses. Unambiguous basecompositions were derived from the exact mass measurements of thecomplementary single-stranded oligonucleotides. Quantitative results areobtained by comparing the peak heights with an internal PCR calibrationstandard present in every PCR well at 500 molecules per well.Calibration methods are commonly owned and disclosed in U.S. ProvisionalPatent Application Ser. No. 60/545,425 which is incorporated herein byreference in entirety.

Example 5 De Novo Determination of Base Composition of AmplificationProducts using Molecular Mass Modified Deoxynucleotide Triphosphates

Because the molecular masses of the four natural nucleobases have arelatively narrow molecular mass range (A=313.058, G=329.052, C=289.046,T=304.046—See Table 4), a persistent source of ambiguity in assignmentof base composition can occur as follows: two nucleic acid strandshaving different base composition may have a difference of about 1 Dawhen the base composition difference between the two strands is G⇄A(−15.994) combined with C⇄T (+15.000). For example, one 99-mer nucleicacid strand having a base composition of A₂₇G₃₀C₂₁T₂₁ has a theoreticalmolecular mass of 30779.058 while another 99-mer nucleic acid strandhaving a base composition of A₂₆G₃₁C₂₂T₂₀ has a theoretical molecularmass of 30780.052. A 1 Da difference in molecular mass may be within theexperimental error of a molecular mass measurement and thus, therelatively narrow molecular mass range of the four natural nucleobasesimposes an uncertainty factor.

The present invention provides for a means for removing this theoretical1 Da uncertainty factor through amplification of a nucleic acid with onemass-tagged nucleobase and three natural nucleobases. The term“nucleobase” as used herein is synonymous with other terms in use in theart including “nucleotide,” “deoxynucleotide,” “nucleotide residue,”“deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP).

Addition of significant mass to one of the 4 nucleobases (dNTPs) in anamplification reaction, or in the primers themselves, will result in asignificant difference in mass of the resulting amplification product(significantly greater than 1 Da) arising from ambiguities arising fromthe G⇄A combined with C ⇄T event (Table 4). Thus, the same the G⇄A(−15.994) event combined with 5-Iodo-C⇄T (−110.900) event would resultin a molecular mass difference of 126.894. If the molecular mass of thebase composition A₂₇G₃₀ 5-Iodo-C₂₁T₂₁ (33422.958) is compared withA₂₆G₃₁5-Iodo-C₂₂T₂₀, (33549.852) the theoretical molecular massdifference is +126.894. The experimental error of a molecular massmeasurement is not significant with regard to this molecular massdifference. Furthermore, the only base composition consistent with ameasured molecular mass of the 99-mer nucleic acid isA₂₇G₃₀5-Iodo-C₂₁T₂₁. In contrast, the analogous amplification withoutthe mass tag has 18 possible base compositions.

TABLE 4 Molecular Masses of Natural Nucleobases and the Mass-ModifiedNucleobase 5-Iodo-C and Molecular Mass Differences Resulting fromTransitions Nucleobase Molecular Mass Transition Molecular Mass A313.058 A-->T −9.012 A 313.058 A-->C −24.012 A 313.058 A-->5-Iodo-C101.888 A 313.058 A-->G 15.994 T 304.046 T-->A 9.012 T 304.046 T-->C−15.000 T 304.046 T-->5-Iodo-C 110.900 T 304.046 T-->G 25.006 C 289.046C-->A 24.012 C 289.046 C-->T 15.000 C 289.046 C-->G 40.006 5-Iodo-C414.946 5-Iodo-C-->A −101.888 5-Iodo-C 414.946 5-Iodo-C-->T −110.9005-Iodo-C 414.946 5-Iodo-C-->G −85.894 G 329.052 G-->A −15.994 G 329.052G-->T −25.006 G 329.052 G-->C −40.006 G 329.052 G-->5-Iodo-C 85.894

Mass spectra of bioagent-identifying amplicons were analyzedindependently using a maximum-likelihood processor, such as is widelyused in radar signal processing. This processor, referred to as GenX,first makes maximum likelihood estimates of the input to the massspectrometer for each primer by running matched filters for each basecomposition aggregate on the input data. This includes the GenX responseto a calibrant for each primer.

The algorithm emphasizes performance predictions culminating inprobability-of-detection versus probability-of-false-alarm plots forconditions involving complex backgrounds of naturally occurringorganisms and environmental contaminants. Matched filters consist of apriori expectations of signal values given the set of primers used foreach of the bioagents. A genomic sequence database is used to define themass base count matched filters. The database contains the sequences ofknown bacterial bioagents and includes threat organisms as well asbenign background organisms. The latter is used to estimate and subtractthe spectral signature produced by the background organisms. A maximumlikelihood detection of known background organisms is implemented usingmatched filters and a running-sum estimate of the noise covariance.Background signal strengths are estimated and used along with thematched filters to form signatures which are then subtracted. Themaximum likelihood process is applied to this “cleaned up” data in asimilar manner employing matched filters for the organisms and arunning-sum estimate of the noise-covariance for the cleaned up data.

The amplitudes of all base compositions of bioagent-identifyingamplicons for each primer are calibrated and a final maximum likelihoodamplitude estimate per organism is made based upon the multiple singleprimer estimates. Models of all system noise are factored into thistwo-stage maximum likelihood calculation. The processor reports thenumber of molecules of each base composition contained in the spectra.The quantity of amplification product corresponding to the appropriateprimer set is reported as well as the quantities of primers remainingupon completion of the amplification reaction.

Base count blurring can be carried out as follows. “Electronic PCR” canbe conducted on nucleotide sequences of the desired bioagents to obtainthe different expected base counts that could be obtained for eachprimer pair. See for example, ncbi.nlm.nih.gov/sutils/e-pcr/; Schuler,Genome Res. 7:541-50, 1997. In one illustrative embodiment, one or morespreadsheets, such as Microsoft Excel workbooks contain a plurality ofworksheets. First in this example, there is a worksheet with a namesimilar to the workbook name; this worksheet contains the raw electronicPCR data. Second, there is a worksheet named “filtered bioagents basecount” that contains bioagent name and base count; there is a separaterecord for each strain after removing sequences that are not identifiedwith a genus and species and removing all sequences for bioagents withless than 10 strains. Third, there is a worksheet, “Sheet1” thatcontains the frequency of substitutions, insertions, or deletions forthis primer pair. This data is generated by first creating a pivot tablefrom the data in the “filtered bioagents base count” worksheet and thenexecuting an Excel VBA macro. The macro creates a table of differencesin base counts for bioagents of the same species, but different strains.One of ordinary skill in the art may understand additional pathways forobtaining similar table differences without undo experimentation.

Application of an exemplary script, involves the user defining athreshold that specifies the fraction of the strains that arerepresented by the reference set of base counts for each bioagent. Thereference set of base counts for each bioagent may contain as manydifferent base counts as are needed to meet or exceed the threshold. Theset of reference base counts is defined by taking the most abundantstrain's base type composition and adding it to the reference set andthen the next most abundant strain's base type composition is addeduntil the threshold is met or exceeded. The current set of data wasobtained using a threshold of 55%, which was obtained empirically.

For each base count not included in the reference base count set forthat bioagent, the script then proceeds to determine the manner in whichthe current base count differs from each of the base counts in thereference set. This difference may be represented as a combination ofsubstitutions, Si=Xi, and insertions, Ii=Yi, or deletions, Di=Zi. Ifthere is more than one reference base count, then the reporteddifference is chosen using rules that aim to minimize the number ofchanges and, in instances with the same number of changes, minimize thenumber of insertions or deletions. Therefore, the primary rule is toidentify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g.,one insertion rather than two substitutions. If there are two or moredifferences with the minimum sum, then the one that will be reported isthe one that contains the most substitutions.

Differences between a base count and a reference composition arecategorized as one, two, or more substitutions, one, two, or moreinsertions, one, two, or more deletions, and combinations ofsubstitutions and insertions or deletions. The different classes ofnucleobase changes and their probabilities of occurrence have beendelineated in U.S. Patent Application Publication No. 2004209260 (U.S.application Ser. No. 10/418,514) which is incorporated herein byreference in entirety.

Example 6 Use of Broad Range Survey and Division Wide Primer Pairs forIdentification of Bacteria in an Epidemic Surveillance Investigation

This investigation employed a set of 16 primer pairs which is hereindesignated the “surveillance primer set” and comprises broad rangesurvey primer pairs, division wide primer pairs and a single Bacilluslade primer pair. The surveillance primer set is shown in Table 5 andconsists of primer pairs originally listed in Table 2. This surveillanceset comprises primers with T modifications (note TMOD designation inprimer names) which constitutes a functional improvement with regard toprevention of non-templated adenylation (vide supra) relative tooriginally selected primers which are displayed below in the same row.Primer pair 449 (non-T modified) has been modified twice. Itspredecessors are primer pairs 70 and 357, displayed below in the samerow. Primer pair 360 has also been modified twice and its predecessorsare primer pairs 17 and 118.

TABLE 5 Bacterial Primer Pairs of the Surveillance Primer Set ForwardReverse Primer Primer Primer Pair (SEQ ID (SEQ ID No. Forward PrimerName NO:) Reverse Primer Name NO:) Target Gene 346 16S_EC_713_732_TMOD_F202 16S_EC_789_809_TMOD_R 1110 16S rRNA 10 16S_EC_713_732_F 2116S_EC_789_809 798 16S rRNA 347 16S_EC_785_806_TMOD_F 56016S_EC_880_897_TMOD_R 1278 16S rRNA 11 16S_EC_785_806_F 11816S_EC_880_897_R 830 16S rRNA 348 16S_EC_960_981_TMOD_F 70616S_EC_1054_1073_TMOD_R 895 16S rRNA 14 16S_EC_960_981_F 67216S_EC_1054_1073_R 735 16S rRNA 349 23S_EC_1826_1843_TMOD_F 40123S_EC_1906_1924_TMOD_R 1156 23S rRNA 16 23S_EC_1826_1843_F 8023S_EC_1906_1924_R 805 23S rRNA 352 INFB_EC_1365_1393_TMOD_F 687INFB_EC_1439_1467_TMOD_R 1411 infB 34 INFB_EC_1365_1393_F 524INFB_EC_1439_1467_R 1248 infB 354 RPOC_EC_2218_2241_TMOD_F 405RPOC_EC_2313_2337_TMOD_R 1072 rpoC 52 RPOC_EC_2218_2241_F 81RPOC_EC_2313_2337_R 790 rpoC 355 SSPE_BA_115_137_TMOD_F 255SSPE_BA_197_222_TMOD_R 1402 sspE 58 SSPE_BA_115_137_F 45SSPE_BA_197_222_R 1201 sspE 356 RPLB_EC_650_679_TMOD_F 232RPLB_EC_739_762_TMOD_R 592 rplB 66 RPLB_EC_650_679_F 98RPLB_EC_739_762_R 999 rplB 358 VALS_EC_1105_1124_TMOD_F 385VALS_EC_1195_1218_TMOD_R 1093 valS 71 VALS_EC_1105_1124_F 77VALS_EC_1195_1218_R 795 valS 359 RPOB_EC_1845_1866_TMOD_F 659RPOB_EC_1909_1929_TMOD_R 1250 rpoB 72 RPOB_EC_1845_1866_F 233RPOB_EC_1909_1929_R 825 rpoB 360 23S_EC_2646_2667_TMOD_F 40923S_EC_2745_2765_TMOD_R 1434 23S rRNA 118 23S_EC_2646_2667_F 8423S_EC_2745_2765_R 1389 23S rRNA 17 23S_EC_2645_2669_F 40823S_EC_2744_2761_R 1252 23S rRNA 361 16S_EC_1090_1111_2_TMOD_F 69716S_EC_1175_1196_TMOD_R 1398 16S rRNA 3 16S_EC_1090_1111_2_F 65116S_EC_1175_1196_R 1159 16S rRNA 362 RPOB_EC_3799_3821_TMOD_F 581RPOB_EC_3862_3888_TMOD_R 1325 rpoB 289 RPOB_EC_3799_3821_F 124RPOB_EC_3862_3888_R 840 rpoB 363 RPOC_EC_2146_2174_TMOD_F 284RPOC_EC_2227_2245_TMOD_R 898 rpoC 290 RPOC_EC_2146_2174_F 52RPOC_EC_2227_2245_R 736 rpoC 367 TUFB_EC_957_979_TMOD_F 308TUFB_EC_1034_1058_TMOD_R 1276 tufB 293 TUFB_EC_957_979_F 55TUFB_EC_1034_1058_R 829 tufB 449 RPLB_EC_690_710_F 309 RPLB_EC_737_758_R1336 rplB 357 RPLB_EC_688_710_TMOD_F 296 RPLB_EC_736_757_TMOD_R 1337rplB 67 RPLB_EC_688_710_F 54 RPLB_EC_736_757_R 842 rplB

The 16 primer pairs of the surveillance set are used to produce bioagentidentifying amplicons whose base compositions are sufficiently differentamongst all known bacteria at the species level to identify, at areasonable confidence level, any given bacterium at the species level.As shown in Tables 6A-E, common respiratory bacterial pathogens can bedistinguished by the base compositions of bioagent identifying ampliconsobtained using the 16 primer pairs of the surveillance set. In somecases, triangulation identification improves the confidence level forspecies assignment. For example, nucleic acid from Streptococcuspyogenes can be amplified by nine of the sixteen surveillance primerpairs and Streptococcus pneumoniae can be amplified by ten of thesixteen surveillance primer pairs. The base compositions of the bioagentidentifying amplicons are identical for only one of the analogousbioagent identifying amplicons and differ in all of the remaininganalogous bioagent identifying amplicons by up to four bases perbioagent identifying amplicon. The resolving power of the surveillanceset was confirmed by determination of base compositions for 120 isolatesof respiratory pathogens representing 70 different bacterial species andthe results indicated that natural variations (usually only one or twobase substitutions per bioagent identifying amplicon) amongst multipleisolates of the same species did not prevent correct identification ofmajor pathogenic organisms at the species level.

Bacillus anthracis is a well known biological warfare agent which hasemerged in domestic terrorism in recent years. Since it was envisionedto produce bioagent identifying amplicons for identification of Bacillusanthracis, additional drill-down analysis primers were designed totarget genes present on virulence plasmids of Bacillus anthracis so thatadditional confidence could be reached in positive identification ofthis pathogenic organism. Three drill-down analysis primers weredesigned and are listed in Tables 2 and 6. In Table 6, the drill-downset comprises primers with T modifications (note TMOD designation inprimer names) which constitutes a functional improvement with regard toprevention of non-templated adenylation (vide supra) relative tooriginally selected primers which are displayed below in the same row.

TABLE 6 Drill-Down Primer Pairs for Confirmation of Identification ofBacillus anthracis Forward Reverse Primer Primer Primer Pair (SEQ ID(SEQ ID No. Forward Primer Name NO:) Reverse Primer Name NO:) TargetGene 350 CAPC_BA_274_303_TMOD_F 476 CAPC_BA_349_376_TMOD_R 1314 capC 24CAPC_BA_274_303_F 109 CAPC_BA_349_376_R 837 capC 351CYA_BA_1353_1379_TMOD_F 355 CYA_BA_1448_1467_TMOD_R 1423 cyA 30CYA_BA_1353_1379_F 64 CYA_BA_1448_1467_R 1342 cyA 353LEF_BA_756_781_TMOD_F 220 LEF_BA_843_872_TMOD_R 1394 lef 37LEF_BA_756_781_F 26 LEF_BA_843_872_R 1135 lef

Phylogenetic coverage of bacterial space of the sixteen surveillanceprimers of Table 5 and the three Bacillus anthracis drill-down primersof Table 6 is shown in FIG. 3 which lists common pathogenic bacteria.FIG. 3 is not meant to be comprehensive in illustrating all speciesidentified by the primers. Only pathogenic bacteria are listed asrepresentative examples of the bacterial species that can be identifiedby the primers and methods of the present invention. Nucleic acid ofgroups of bacteria enclosed within the polygons of FIG. 3 can beamplified to obtain bioagent identifying amplicons using the primer pairnumbers listed in the upper right hand corner of each polygon. Primercoverage for polygons within polygons is additive. As an illustrativeexample, bioagent identifying amplicons can be obtained for Chlamydiatrachomatis by amplification with, for example, primer pairs 346-349,360 and 361, but not with any of the remaining primers of thesurveillance primer set. On the other hand, bioagent identifyingamplicons can be obtained from nucleic acid originating from Bacillusanthracis (located within 5 successive polygons) using, for example, anyof the following primer pairs: 346-349, 360, 361 (base polygon), 356,449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350,351 and 353 (fifth polygon). Multiple coverage of a given organism withmultiple primers provides for increased confidence level inidentification of the organism as a result of enabling broadtriangulation identification.

In Tables 7A-E, base compositions of respiratory pathogens for primertarget regions are shown. Two entries in a cell, represent variation inribosomal DNA operons. The most predominant base composition is shownfirst and the minor (frequently a single operon) is indicated by anasterisk (*). Entries with NO DATA mean that the primer would not beexpected to prime this species due to mismatches between the primer andtarget region, as determined by theoretical PCR.

TABLE 7A Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 346, 347 and 348Primer 346 Primer 347 Primer 348 Organism Strain [A G C T] [A G C T] [AG C T] Klebsiella MGH78578 [29 32 25 13] [23 38 28 26] [26 32 28 30]pneumoniae [29 31 25 13]* [23 37 28 26]* [26 31 28 30]* Yersinia pestisCO-92 Biovar [29 32 25 13] [22 39 28 26] [29 30 28 29] Orientalis [30 3027 29]* Yersinia pestis KIM5 P12 (Biovar [29 32 25 13] [22 39 28 26] [2930 28 29] Mediaevalis) Yersinia pestis 91001 [29 32 25 13] [22 39 28 26][29 30 28 29] [30 30 27 29]* Haemophilus KW20 [28 31 23 17] [24 37 2527] [29 30 28 29] influenzae Pseudomonas PAO1 [30 31 23 15] [26 36 2924] [26 32 29 29] aeruginosa [27 36 29 23]* Pseudomonas Pf0-1 [30 31 2315] [26 35 29 25] [28 31 28 29] fluorescens Pseudomonas KT2440 [30 31 2315] [28 33 27 27] [27 32 29 28] putida Legionella Philadelphia-1 [30 3024 15] [33 33 23 27] [29 28 28 31] pneumophila Francisella schu 4 [32 2922 16] [28 38 26 26] [25 32 28 31] tularensis Bordetella Tohama I [30 2924 16] [23 37 30 24] [30 32 30 26] pertussis Burkholderia J2315 [29 2927 14] [27 32 26 29] [27 36 31 24] cepacia [20 42 35 19]* BurkholderiaK96243 [29 29 27 14] [27 32 26 29] [27 36 31 24] pseudomallei NeisseriaFA 1090, ATCC [29 28 24 18] [27 34 26 28] [24 36 29 27] gonorrhoeae700825 Neisseria MC58 (serogroup B) [29 28 26 16] [27 34 27 27] [25 3530 26] meningitidis Neisseria serogroup C, FAM18 [29 28 26 16] [27 34 2727] [25 35 30 26] meningitidis Neisseria Z2491 (serogroup A) [29 28 2616] [27 34 27 27] [25 35 30 26] meningitidis Chlamydophila TW-183 [31 2722 19] NO DATA [32 27 27 29] pneumoniae Chlamydophila AR39 [31 27 22 19]NO DATA [32 27 27 29] pneumoniae Chlamydophila CWL029 [31 27 22 19] NODATA [32 27 27 29] pneumoniae Chlamydophila J138 [31 27 22 19] NO DATA[32 27 27 29] pneumoniae Corynebacterium NCTC13129 [29 34 21 15] [22 3831 25] [22 33 25 34] diphtheriae Mycobacterium k10 [27 36 21 15] [22 3730 28] [21 36 27 30] avium Mycobacterium 104 [27 36 21 15] [22 37 30 28][21 36 27 30] avium Mycobacterium CSU#93 [27 36 21 15] [22 37 30 28] [2136 27 30] tuberculosis Mycobacterium CDC 1551 [27 36 21 15] [22 37 3028] [21 36 27 30] tuberculosis Mycobacterium H37Rv (lab strain) [27 3621 15] [22 37 30 28] [21 36 27 30] tuberculosis Mycoplasma M129 [31 2919 20] NO DATA NO DATA pneumoniae Staphylococcus MRSA252 [27 30 21 21][25 35 30 26] [30 29 30 29] aureus [29 31 30 29]* Staphylococcus MSSA476[27 30 21 21] [25 35 30 26] [30 29 30 29] aureus [30 29 29 30]*Staphylococcus COL [27 30 21 21] [25 35 30 26] [30 29 30 29] aureus [3029 29 30]* Staphylococcus Mu50 [27 30 21 21] [25 35 30 26] [30 29 30 29]aureus [30 29 29 30]* Staphylococcus MW2 [27 30 21 21] [25 35 30 26] [3029 30 29] aureus [30 29 29 30]* Staphylococcus N315 [27 30 21 21] [25 3530 26] [30 29 30 29] aureus [30 29 29 30]* Staphylococcus NCTC 8325 [2730 21 21] [25 35 30 26] [30 29 30 29] aureus [25 35 31 26]* [30 29 2930] Streptococcus NEM316 [26 32 23 18] [24 36 31 25] [25 32 29 30]agalactiae [24 36 30 26]* Streptococcus NC_002955 [26 32 23 18] [23 3731 25] [29 30 25 32] equi Streptococcus MGAS8232 [26 32 23 18] [24 37 3025] [25 31 29 31] pyogenes Streptococcus MGAS315 [26 32 23 18] [24 37 3025] [25 31 29 31] pyogenes Streptococcus SSI-1 [26 32 23 18] [24 37 3025] [25 31 29 31] pyogenes Streptococcus MGAS10394 [26 32 23 18] [24 3730 25] [25 31 29 31] pyogenes Streptococcus Manfredo (M5) [26 32 23 18][24 37 30 25] [25 31 29 31] pyogenes Streptococcus SF370 (M1) [26 32 2318] [24 37 30 25] [25 31 29 31] pyogenes Streptococcus 670 [26 32 23 18][25 35 28 28] [25 32 29 30] pneumoniae Streptococcus R6 [26 32 23 18][25 35 28 28] [25 32 29 30] pneumoniae Streptococcus TIGR4 [26 32 23 18][25 35 28 28] [25 32 30 29] pneumoniae Streptococcus NCTC7868 [25 33 2318] [24 36 31 25] [25 31 29 31] gordonii Streptococcus NCTC 12261 [26 3223 18] [25 35 30 26] [25 32 29 30] mitis [24 31 35 29]* StreptococcusUA159 [24 32 24 19] [25 37 30 24] [28 31 26 31] mutans

TABLE 7B Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 349, 360, and356 Primer 349 Primer 360 Primer 356 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 [25 31 25 22] [33 37 25 27] NO DATApneumoniae Yersinia pestis CO-92 Biovar [25 31 27 20] [34 35 25 28] NODATA Orientalis [25 32 26 20]* Yersinia pestis KIM5 P12 (Biovar [25 3127 20] [34 35 25 28] NO DATA Mediaevalis) [25 32 26 20]* Yersinia pestis91001 [25 31 27 20] [34 35 25 28] NO DATA Haemophilus KW20 [28 28 25 20][32 38 25 27] NO DATA influenzae Pseudomonas PAO1 [24 31 26 20] [31 3627 27] NO DATA aeruginosa [31 36 27 28]* Pseudomonas Pf0-1 NO DATA [3037 27 28] NO DATA fluorescens [30 37 27 28] Pseudomonas KT2440 [24 31 2620] [30 37 27 28] NO DATA putida Legionella Philadelphia-1 [23 30 25 23][30 39 29 24] NO DATA pneumophila Francisella schu 4 [26 31 25 19] [3236 27 27] NO DATA tularensis Bordetella Tohama I [21 29 24 18] [33 36 2627] NO DATA pertussis Burkholderia J2315 [23 27 22 20] [31 37 28 26] NODATA cepacia Burkholderia K96243 [23 27 22 20] [31 37 28 26] NO DATApseudomallei Neisseria FA 1090, ATCC 700825 [24 27 24 17] [34 37 25 26]NO DATA gonorrhoeae Neisseria MC58 (serogroup B) [25 27 22 18] [34 37 2526] NO DATA meningitidis Neisseria serogroup C, FAM18 [25 26 23 18] [3437 25 26] NO DATA meningitidis Neisseria Z2491 (serogroup A) [25 26 2318] [34 37 25 26] NO DATA meningitidis Chlamydophila TW-183 [30 28 2718] NO DATA NO DATA pneumoniae Chlamydophila AR39 [30 28 27 18] NO DATANO DATA pneumoniae Chlamydophila CWL029 [30 28 27 18] NO DATA NO DATApneumoniae Chlamydophila J138 [30 28 27 18] NO DATA NO DATA pneumoniaeCorynebacterium NCTC13129 NO DATA [29 40 28 25] NO DATA diphtheriaeMycobacterium k10 NO DATA [33 35 32 22] NO DATA avium Mycobacterium 104NO DATA [33 35 32 22] NO DATA avium Mycobacterium CSU#93 NO DATA [30 3634 22] NO DATA tuberculosis Mycobacterium CDC 1551 NO DATA [30 36 34 22]NO DATA tuberculosis Mycobacterium H37Rv (lab strain) NO DATA [30 36 3422] NO DATA tuberculosis Mycoplasma M129 [28 30 24 19] [34 31 29 28] NODATA pneumoniae Staphylococcus MRSA252 [26 30 25 20] [31 38 24 29] [3330 31 27] aureus Staphylococcus MSSA476 [26 30 25 20] [31 38 24 29] [3330 31 27] aureus Staphylococcus COL [26 30 25 20] [31 38 24 29] [33 3031 27] aureus Staphylococcus Mu50 [26 30 25 20] [31 38 24 29] [33 30 3127] aureus Staphylococcus MW2 [26 30 25 20] [31 38 24 29] [33 30 31 27]aureus Staphylococcus N315 [26 30 25 20] [31 38 24 29] [33 30 31 27]aureus Staphylococcus NCTC 8325 [26 30 25 20] [31 38 24 29] [33 30 3127] aureus Streptococcus NEM316 [28 31 22 20] [33 37 24 28] [37 30 2826] agalactiae Streptococcus NC_002955 [28 31 23 19] [33 38 24 27] [3731 28 25] equi Streptococcus MGAS8232 [28 31 23 19] [33 37 24 28] [38 3129 23] pyogenes Streptococcus MGAS315 [28 31 23 19] [33 37 24 28] [38 3129 23] pyogenes Streptococcus SSI-1 [28 31 23 19] [33 37 24 28] [38 3129 23] pyogenes Streptococcus MGAS10394 [28 31 23 19] [33 37 24 28] [3831 29 23] pyogenes Streptococcus Manfredo (M5) [28 31 23 19] [33 37 2428] [38 31 29 23] pyogenes Streptococcus SF370 (M1) [28 31 23 19] [33 3724 28] [38 31 29 23] pyogenes [28 31 22 20]* Streptococcus 670 [28 31 2220] [34 36 24 28] [37 30 29 25] pneumoniae Streptococcus R6 [28 31 2220] [34 36 24 28] [37 30 29 25] pneumoniae Streptococcus TIGR4 [28 31 2220] [34 36 24 28] [37 30 29 25] pneumoniae Streptococcus NCTC7868 [28 3223 20] [34 36 24 28] [36 31 29 25] gordonii Streptococcus NCTC 12261 [2831 22 20] [34 36 24 28] [37 30 29 25] mitis [29 30 22 20]* StreptococcusUA159 [26 32 23 22] [34 37 24 27] NO DATA mutans

TABLE 7C Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 449, 354, and352 Primer 449 Primer 354 Primer 352 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 NO DATA [27 33 36 26] NO DATA pneumoniaeYersinia pestis CO-92 Biovar NO DATA [29 31 33 29] [32 28 20 25]Orientalis Yersinia pestis KIM5 P12 (Biovar NO DATA [29 31 33 29] [32 2820 25] Mediaevalis) Yersinia pestis 91001 NO DATA [29 31 33 29] NO DATAHaemophilus KW20 NO DATA [30 29 31 32] NO DATA influenzae PseudomonasPAO1 NO DATA [26 33 39 24] NO DATA aeruginosa Pseudomonas Pf0-1 NO DATA[26 33 34 29] NO DATA fluorescens Pseudomonas KT2440 NO DATA [25 34 3627] NO DATA putida Legionella Philadelphia-1 NO DATA NO DATA NO DATApneumophila Francisella schu 4 NO DATA [33 32 25 32] NO DATA tularensisBordetella Tohama I NO DATA [26 33 39 24] NO DATA pertussis BurkholderiaJ2315 NO DATA [25 37 33 27] NO DATA cepacia Burkholderia K96243 NO DATA[25 37 34 26] NO DATA pseudomallei Neisseria FA 1090, ATCC 700825 [17 2322 10] [29 31 32 30] NO DATA gonorrhoeae Neisseria MC58 (serogroup B) NODATA [29 30 32 31] NO DATA meningitidis Neisseria serogroup C, FAM18 NODATA [29 30 32 31] NO DATA meningitidis Neisseria Z2491 (serogroup A) NODATA [29 30 32 31] NO DATA meningitidis Chlamydophila TW-183 NO DATA NODATA NO DATA pneumoniae Chlamydophila AR39 NO DATA NO DATA NO DATApneumoniae Chlamydophila CWL029 NO DATA NO DATA NO DATA pneumoniaeChlamydophila J138 NO DATA NO DATA NO DATA pneumoniae CorynebacteriumNCTC13129 NO DATA NO DATA NO DATA diphtheriae Mycobacterium k10 NO DATANO DATA NO DATA avium Mycobacterium 104 NO DATA NO DATA NO DATA aviumMycobacterium CSU#93 NO DATA NO DATA NO DATA tuberculosis MycobacteriumCDC 1551 NO DATA NO DATA NO DATA tuberculosis Mycobacterium H37Rv (labstrain) NO DATA NO DATA NO DATA tuberculosis Mycoplasma M129 NO DATA NODATA NO DATA pneumoniae Staphylococcus MRSA252 [17 20 21 17] [30 27 3035] [36 24 19 26] aureus Staphylococcus MSSA476 [17 20 21 17] [30 27 3035] [36 24 19 26] aureus Staphylococcus COL [17 20 21 17] [30 27 30 35][35 24 19 27] aureus Staphylococcus Mu50 [17 20 21 17] [30 27 30 35] [3624 19 26] aureus Staphylococcus MW2 [17 20 21 17] [30 27 30 35] [36 2419 26] aureus Staphylococcus N315 [17 20 21 17] [30 27 30 35] [36 24 1926] aureus Staphylococcus NCTC 8325 [17 20 21 17] [30 27 30 35] [35 2419 27] aureus Streptococcus NEM316 [22 20 19 14] [26 31 27 38] [29 26 2228] agalactiae Streptococcus NC_002955 [22 21 19 13] NO DATA NO DATAequi Streptococcus MGAS8232 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus MGAS315 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus SSI-1 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus MGAS10394 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus Manfredo (M5) [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus SF370 (M1) [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus 670 [22 20 19 14] [25 33 29 35] [30 29 21 25] pneumoniaeStreptococcus R6 [22 20 19 14] [25 33 29 35] [30 29 21 25] pneumoniaeStreptococcus TIGR4 [22 20 19 14] [25 33 29 35] [30 29 21 25] pneumoniaeStreptococcus NCTC7868 [21 21 19 14] NO DATA [29 26 22 28] gordoniiStreptococcus NCTC 12261 [22 20 19 14] [26 30 32 34] NO DATA mitisStreptococcus UA159 NO DATA NO DATA NO DATA mutans

TABLE 7D Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 355, 358, and359 Primer 355 Primer 358 Primer 359 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 NO DATA [24 39 33 20] [25 21 24 17]pneumoniae Yersinia pestis CO-92 Biovar NO DATA [26 34 35 21] [23 23 1922] Orientalis Yersinia pestis KIM5 P12 (Biovar NO DATA [26 34 35 21][23 23 19 22] Mediaevalis) Yersinia pestis 91001 NO DATA [26 34 35 21][23 23 19 22] Haemophilus KW20 NO DATA NO DATA NO DATA influenzaePseudomonas PAO1 NO DATA NO DATA NO DATA aeruginosa Pseudomonas Pf0-1 NODATA NO DATA NO DATA fluorescens Pseudomonas KT2440 NO DATA [21 37 3721] NO DATA putida Legionella Philadelphia-1 NO DATA NO DATA NO DATApneumophila Francisella schu 4 NO DATA NO DATA NO DATA tularensisBordetella Tohama I NO DATA NO DATA NO DATA pertussis Burkholderia J2315NO DATA NO DATA NO DATA cepacia Burkholderia K96243 NO DATA NO DATA NODATA pseudomallei Neisseria FA 1090, ATCC 700825 NO DATA NO DATA NO DATAgonorrhoeae Neisseria MC58 (serogroup B) NO DATA NO DATA NO DATAmeningitidis Neisseria serogroup C, FAM18 NO DATA NO DATA NO DATAmeningitidis Neisseria Z2491 (serogroup A) NO DATA NO DATA NO DATAmeningitidis Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniaeChlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae ChlamydophilaCWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NODATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NODATA diphtheriae Mycobacterium k10 NO DATA NO DATA NO DATA aviumMycobacterium 104 NO DATA NO DATA NO DATA avium Mycobacterium CSU#93 NODATA NO DATA NO DATA tuberculosis Mycobacterium CDC 1551 NO DATA NO DATANO DATA tuberculosis Mycobacterium H37Rv (lab strain) NO DATA NO DATA NODATA tuberculosis Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniaeStaphylococcus MRSA252 NO DATA NO DATA NO DATA aureus StaphylococcusMSSA476 NO DATA NO DATA NO DATA aureus Staphylococcus COL NO DATA NODATA NO DATA aureus Staphylococcus Mu50 NO DATA NO DATA NO DATA aureusStaphylococcus MW2 NO DATA NO DATA NO DATA aureus Staphylococcus N315 NODATA NO DATA NO DATA aureus Staphylococcus NCTC 8325 NO DATA NO DATA NODATA aureus Streptococcus NEM316 NO DATA NO DATA NO DATA agalactiaeStreptococcus NC_002955 NO DATA NO DATA NO DATA equi StreptococcusMGAS8232 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS315 NO DATANO DATA NO DATA pyogenes Streptococcus SSI-1 NO DATA NO DATA NO DATApyogenes Streptococcus MGAS10394 NO DATA NO DATA NO DATA pyogenesStreptococcus Manfredo (M5) NO DATA NO DATA NO DATA pyogenesStreptococcus SF370 (M1) NO DATA NO DATA NO DATA pyogenes Streptococcus670 NO DATA NO DATA NO DATA pneumoniae Streptococcus R6 NO DATA NO DATANO DATA pneumoniae Streptococcus TIGR4 NO DATA NO DATA NO DATApneumoniae Streptococcus NCTC7868 NO DATA NO DATA NO DATA gordoniiStreptococcus NCTC 12261 NO DATA NO DATA NO DATA mitis StreptococcusUA159 NO DATA NO DATA NO DATA mutans

TABLE 7E Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 362, 363, and367 Primer 362 Primer 363 Primer 367 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 [21 33 22 16] [16 34 26 26] NO DATApneumoniae Yersinia pestis CO-92 Biovar [20 34 18 20] NO DATA NO DATAOrientalis Yersinia pestis KIM5 P12 (Biovar [20 34 18 20] NO DATA NODATA Mediaevalis) Yersinia pestis 91001 [20 34 18 20] NO DATA NO DATAHaemophilus KW20 NO DATA NO DATA NO DATA influenzae Pseudomonas PAO1 [1935 21 17] [16 36 28 22] NO DATA aeruginosa Pseudomonas Pf0-1 NO DATA [1835 26 23] NO DATA fluorescens Pseudomonas KT2440 NO DATA [16 35 28 23]NO DATA putida Legionella Philadelphia-1 NO DATA NO DATA NO DATApneumophila Francisella schu 4 NO DATA NO DATA NO DATA tularensisBordetella Tohama I [20 31 24 17] [15 34 32 21] [26 25 34 19] pertussisBurkholderia J2315 [20 33 21 18] [15 36 26 25] [25 27 32 20] cepaciaBurkholderia K96243 [19 34 19 20] [15 37 28 22] [25 27 32 20]pseudomallei Neisseria FA 1090, ATCC 700825 NO DATA NO DATA NO DATAgonorrhoeae Neisseria MC58 (serogroup B) NO DATA NO DATA NO DATAmeningitidis Neisseria serogroup C, FAM18 NO DATA NO DATA NO DATAmeningitidis Neisseria Z2491 (serogroup A) NO DATA NO DATA NO DATAmeningitidis Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniaeChlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae ChlamydophilaCWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NODATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NODATA diphtheriae Mycobacterium k10 [19 34 23 16] NO DATA [24 26 35 19]avium Mycobacterium 104 [19 34 23 16] NO DATA [24 26 35 19] aviumMycobacterium CSU#93 [19 31 25 17] NO DATA [25 25 34 20] tuberculosisMycobacterium CDC 1551 [19 31 24 18] NO DATA [25 25 34 20] tuberculosisMycobacterium H37Rv (lab strain) [19 31 24 18] NO DATA [25 25 34 20]tuberculosis Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniaeStaphylococcus MRSA252 NO DATA NO DATA NO DATA aureus StaphylococcusMSSA476 NO DATA NO DATA NO DATA aureus Staphylococcus COL NO DATA NODATA NO DATA aureus Staphylococcus Mu50 NO DATA NO DATA NO DATA aureusStaphylococcus MW2 NO DATA NO DATA NO DATA aureus Staphylococcus N315 NODATA NO DATA NO DATA aureus Staphylococcus NCTC 8325 NO DATA NO DATA NODATA aureus Streptococcus NEM316 NO DATA NO DATA NO DATA agalactiaeStreptococcus NC_002955 NO DATA NO DATA NO DATA equi StreptococcusMGAS8232 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS315 NO DATANO DATA NO DATA pyogenes Streptococcus SSI-1 NO DATA NO DATA NO DATApyogenes Streptococcus MGAS10394 NO DATA NO DATA NO DATA pyogenesStreptococcus Manfredo (M5) NO DATA NO DATA NO DATA pyogenesStreptococcus SF370 (M1) NO DATA NO DATA NO DATA pyogenes Streptococcus670 NO DATA NO DATA NO DATA pneumoniae Streptococcus R6 [20 30 19 23] NODATA NO DATA pneumoniae Streptococcus TIGR4 [20 30 19 23] NO DATA NODATA pneumoniae Streptococcus NCTC7868 NO DATA NO DATA NO DATA gordoniiStreptococcus NCTC 12261 NO DATA NO DATA NO DATA mitis StreptococcusUA159 NO DATA NO DATA NO DATA mutans

Four sets of throat samples from military recruits at different militaryfacilities taken at different time points were analyzed using theprimers of the present invention. The first set was collected at amilitary training center from Nov. 1 to Dec. 20, 2002 during one of themost severe outbreaks of pneumonia associated with group A Streptococcusin the United States since 1968. During this outbreak, fifty-one throatswabs were taken from both healthy and hospitalized recruits and platedon blood agar for selection of putative group A Streptococcus colonies.A second set of 15 original patient specimens was taken during theheight of this group A Streptococcus—associated respiratory diseaseoutbreak. The third set were historical samples, including twenty-sevenisolates of group A Streptococcus, from disease outbreaks at this andother military training facilities during previous years. The fourth setof samples was collected from five geographically separated militaryfacilities in the continental U.S. in the winter immediately followingthe severe November/December 2002 outbreak.

Pure colonies isolated from group A Streptococcus-selective media fromall four collection periods were analyzed with the surveillance primerset. All samples showed base compositions that precisely matched thefour completely sequenced strains of Streptococcus pyogenes. Shown inFIG. 4 is a 3D diagram of base composition (axes A, G and C) of bioagentidentifying amplicons obtained with primer pair number 14 (a precursorof primer pair number 348 which targets 16S rRNA). The diagram indicatesthat the experimentally determined base compositions of the clinicalsamples closely match the base compositions expected for Streptococcuspyogenes and are distinct from the expected base compositions of otherorganisms.

In addition to the identification of Streptococcus pyogenes, otherpotentially pathogenic organisms were identified concurrently. Massspectral analysis of a sample whose nucleic acid was amplified by primerpair number 349 (SEQ ID NOs: 401:1156) exhibited signals of bioagentidentifying amplicons with molecular masses that were found tocorrespond to analogous base compositions of bioagent identifyingamplicons of Streptococcus pyogenes (A27 G32 C24 Ti 8), Neisseriameningitidis (A25 G27 C22 T18 Haemophilus influenzae (A28 G28 C25 T20)(see FIG. 5 and Table 7B). These organisms were present in a ratio of4:5:20 as determined by comparison of peak heights with peak height ofan internal PCR calibration standard as described in commonly owned U.S.Patent Application Ser. No: 60/545,425 which is incorporated herein byreference in its entirety.

Since certain division-wide primers that target housekeeping genes aredesigned to provide coverage of specific divisions of bacteria toincrease the confidence level for identification of bacterial species,they are not expected to yield bioagent identifying amplicons fororganisms outside of the specific divisions. For example, primer pairnumber 356 (SEQ ID NOs: 449:1380) primarily amplifies the nucleic acidof members of the classes Bacilli and Clostridia and is not expected toamplify proteobacteria such as Neisseria meningitidis and Haemophilusinfluenzae. As expected, analysis of the mass spectrum of amplificationproducts obtained with primer pair number 356 does not indicate thepresence of Neisseria meningitidis and Haemophilus influenzae but doesindicate the presence of Streptococcus pyogenes (FIGS. 3 and 6, Table7B). Thus, these primers or types of primers can confirm the absence ofparticular bioagents from a sample.

The 15 throat swabs from military recruits were found to contain arelatively small set of microbes in high abundance. The most common wereHaemophilus influenza, Neisseria meningitides, and Streptococcuspyogenes. Staphylococcus epidermidis, Moraxella cattarhalis,Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus werepresent in fewer samples. An equal number of samples from healthyvolunteers from three different geographic locations, were identicallyanalyzed. Results indicated that the healthy volunteers have bacterialflora dominated by multiple, commensal non-beta-hemolytic Streptococcalspecies, including the viridans group streptococci (S. parasangunis, S.vestibularis, S. mitis, S. oralis and S. pneumoniae; data not shown),and none of the organisms found in the military recruits were found inthe healthy controls at concentrations detectable by mass spectrometry.Thus, the military recruits in the midst of a respiratory diseaseoutbreak had a dramatically different microbial population than thatexperienced by the general population in the absence of epidemicdisease.

Example 7 Triangulation Genotyping Analysis for Determination ofemm-Type of Streptococcus pyogenes in Epidemic Surveillance

As a continuation of the epidemic surveillance investigation of Example6, determination of sub-species characteristics (genotyping) ofStreptococcus pyogenes, was carried out based on a strategy thatgenerates strain-specific signatures according to the rationale ofMulti-Locus Sequence Typing (MLST). In classic MLST analysis, internalfragments of several housekeeping genes are amplified and sequenced(Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classicMLST analysis, internal fragments of several housekeeping genes areamplified and sequenced. In the present investigation, bioagentidentifying amplicons from housekeeping genes were produced usingdrill-down primers and analyzed by mass spectrometry. Since massspectral analysis results in molecular mass, from which base compositioncan be determined, the challenge was to determine whether resolution ofemm classification of strains of Streptococcus pyogenes could bedetermined.

For the purpose of development of a triangulation genotyping assay, analignment was constructed of concatenated alleles of seven MLSThousekeeping genes (glucose kinase (gki), glutamine transporter protein(gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS),xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyltransferase (yqiL)) from each of the 212 previously emm-typed strains ofStreptococcus pyogenes. From this alignment, the number and location ofprimer pairs that would maximize strain identification via basecomposition was determined. As a result, 6 primer pairs were chosen asstandard drill-down primers for determination of emm-type ofStreptococcus pyogenes. These six primer pairs are displayed in Table 8.This drill-down set comprises primers with T modifications (note TMODdesignation in primer names) which constitutes a functional improvementwith regard to prevention of non-templated adenylation (vide supra)relative to originally selected primers which are displayed below in thesame row.

TABLE 8 Triangulation Genotyping Analysis Primer Pairs for Group AStreptococcus Drill-Down Primer Forward Primer Reverse Primer Pair No.Forward Primer Name (SEQ ID NO:) Reverse Primer Name (SEQ ID NO:) TargetGene 442 SP101_SPET11_358_387_TMOD_F 588 SP101_SPET11_448_473_TMOD_R 998gki 80 SP101_SPET11_358_387_F 126 SP101_SPET11_448_473_TMOD_R 766 gki443 SP101_SPET11_600_629_TMOD_F 348 SP101_SPET11_686_714_TMOD_R 1018 gtr81 SP101_SPET11_600_629_F 62 SP101_SPET11_686_714_R 772 gtr 426SP101_SPET11_1314_1336_TMOD_F 363 SP101_SPET11_1403_1431_TMOD_R 849 murI86 SP101_SPET11_1314_1336_F 68 SP101_SPET11_1403_1431_R 711 murI 430SP101_SPET11_1807_1835_TMOD_F 235 SP101_SPET11_1901_1927_TMOD_R 1439mutS 90 SP101_SPET11_1807_1835_F 33 SP101_SPET11_1901_1927_R 1412 mutS438 SP101_SPET11_3075_3103_TMOD_F 473 SP101_SPET11_3168_3196_TMOD_R 875xpt 96 SP101_SPET11_3075_3103_F 108 SP101_SPET11_3168_3196_R 715 xpt 441SP101_SPET11_3511_3535_TMOD_F 531 SP101_SPET11_3605_3629_TMOD_R 1294yqiL 98 SP101_SPET11_3511_3535_F 116 SP101_SPET11_3605_3629_R 832 yqiL

The primers of Table 8 were used to produce bioagent identifyingamplicons from nucleic acid present in the clinical samples. Thebioagent identifying amplicons which were subsequently analyzed by massspectrometry and base compositions corresponding to the molecular masseswere calculated.

Of the 51 samples taken during the peak of the November/December 2002epidemic (Table 9A-C rows 1-3), all except three samples were found torepresent emm3, a Group A Streptococcus genotype previously associatedwith high respiratory virulence. The three outliers were from samplesobtained from healthy individuals and probably represent non-epidemicstrains. Archived samples (Tables 9A-C rows 5-13) from historicalcollections showed a greater heterogeneity of base compositions and emmtypes as would be expected from different epidemics occurring atdifferent places and dates. The results of the mass spectrometryanalysis and emm gene sequencing were found to be concordant for theepidemic and historical samples.

TABLE 9A Base Composition Analysis of Bioagent Identifying Amplicons ofGroup A Streptococcus samples from Six Military Installations Obtainedwith Primer Pair Nos. 426 and 430 emm-type by murI mutS # of Massemm-Gene Location (Primer Pair (Primer Pair Instances SpectrometrySequencing (sample) Year No. 426) No. 430) 48  3  3 MCRD San 2002 A39G25 C20 T34 A38 G27 C23 T33 2 6  6 Diego A40 G24 C20 T34 A38 G27 C23 T331 28  28 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 15  3 ND A39 G25 C20T34 A38 G27 C23 T33 6 3  3 NHRC San 2003 A39 G25 C20 T34 A38 G27 C23 T333 5, 58  5 Diego- A40 G24 C20 T34 A38 G27 C23 T33 6 6  6 Archive A40 G24C20 T34 A38 G27 C23 T33 1 11  11 (Cultured) A39 G25 C20 T34 A38 G27 C23T33 3 12  12 A40 G24 C20 T34 A38 G26 C24 T33 1 22  22 A39 G25 C20 T34A38 G27 C23 T33 3 25, 75  75 A39 G25 C20 T34 A38 G27 C23 T33 4 44/61,82, 9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 53, 91 91 A39 G25 C20 T34A38 G27 C23 T33 1 2  2 Ft. 2003 A39 G25 C20 T34 A38 G27 C24 T32 2 3  3Leonard A39 G25 C20 T34 A38 G27 C23 T33 1 4  4 Wood A39 G25 C20 T34 A38G27 C23 T33 1 6  6 (Cultured) A40 G24 C20 T34 A38 G27 C23 T33 11  25 or75 75 A39 G25 C20 T34 A38 G27 C23 T33 1 25, 75, 33, 75 A39 G25 C20 T34A38 G27 C23 T33 34, 4, 52, 84 1 44/61 or 82 44/61 A40 G24 C20 T34 A38G26 C24 T33 or 9 2 5 or 58  5 A40 G24 C20 T34 A38 G27 C23 T33 3 1  1 Ft.Sill 2003 A40 G24 C20 T34 A38 G27 C23 T33 2 3  3 (Cultured) A39 G25 C20T34 A38 G27 C23 T33 1 4  4 A39 G25 C20 T34 A38 G27 C23 T33 1 28  28 A39G25 C20 T34 A38 G27 C23 T33 1 3  3 Ft. 2003 A39 G25 C20 T34 A38 G27 C23T33 1 4  4 Benning A39 G25 C20 T34 A38 G27 C23 T33 3 6  6 (Cultured) A40G24 C20 T34 A38 G27 C23 T33 1 11  11 A39 G25 C20 T34 A38 G27 C23 T33 113   94** A40 G24 C20 T34 A38 G27 C23 T33 1 44/61 or 82 82 A40 G24 C20T34 A38 G26 C24 T33 or 9 1 5 or 58 58 A40 G24 C20 T34 A38 G27 C23 T33 178 or 89 89 A39 G25 C20 T34 A38 G27 C23 T33 2 5 or 58 ND Lackland 2003A40 G24 C20 T34 A38 G27 C23 T33 1 2 AFB A39 G25 C20 T34 A38 G27 C24 T321 81 or 90 (Throat A40 G24 C20 T34 A38 G27 C23 T33 1 78  Swabs) A38 G26C20 T34 A38 G27 C23 T33   3*** No detection No detection No detection 73 ND MCRD San 2002 A39 G25 C20 T34 A38 G27 C23 T33 1 3 ND Diego Nodetection A38 G27 C23 T33 1 3 ND (Throat No detection No detection 1 3ND Swabs) No detection No detection 2 3 ND No detection A38 G27 C23 T333 No detection ND No detection No detection

TABLE 9B Base Composition Analysis of Bioagent Identifying Amplicons ofGroup A Streptococcus samples from Six Military Installations Obtainedwith Primer Pair Nos. 438 and 441 emm-type by xpt yqiL # of Massemm-Gene Location (Primer Pair (Primer Pair Instances SpectrometrySequencing (sample) Year No. 438) No. 441) 48  3  3 MCRD San 2002 A30G36 C20 T36 A40 G29 C19 T31 2 6  6 Diego A30 G36 C20 T36 A40 G29 C19 T311 28  28 (Cultured) A30 G36 C20 T36 A41 G28 C18 T32 15  3 ND A30 G36 C20T36 A40 G29 C19 T31 6 3  3 NHRC San 2003 A30 G36 C20 T36 A40 G29 C19 T313 5, 58  5 Diego- A30 G36 C20 T36 A40 G29 C19 T31 6 6  6 Archive A30 G36C20 T36 A40 G29 C19 T31 1 11  11 (Cultured) A30 G36 C20 T36 A40 G29 C19T31 3 12  12 A30 G36 C19 T37 A40 G29 C19 T31 1 22  22 A30 G36 C20 T36A40 G29 C19 T31 3 25, 75 75 A30 G36 C20 T36 A40 G29 C19 T31 4 44/61, 82,9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 53, 91 91 A30 G36 C19 T37 A40G29 C19 T31 1 2  2 Ft. 2003 A30 G36 C20 T36 A40 G29 C19 T31 2 3  3Leonard A30 G36 C20 T36 A40 G29 C19 T31 1 4  4 Wood A30 G36 C19 T37 A41G28 C19 T31 1 6  6 (Cultured) A30 G36 C20 T36 A40 G29 C19 T31 11  25 or75 75 A30 G36 C20 T36 A40 G29 C19 T31 1 25, 75, 33, 75 A30 G36 C19 T37A40 G29 C19 T31 34, 4, 52, 84 1 44/61 or 82 44/61 A30 G36 C20 T36 A41G28 C19 T31 or 9 2 5 or 58  5 A30 G36 C20 T36 A40 G29 C19 T31 3 1  1 Ft.Sill 2003 A30 G36 C19 T37 A40 G29 C19 T31 2 3  3 (Cultured) A30 G36 C20T36 A40 G29 C19 T31 1 4  4 A30 G36 C19 T37 A41 G28 C19 T31 1 28  28 A30G36 C20 T36 A41 G28 C18 T32 1 3  3 Ft. 2003 A30 G36 C20 T36 A40 G29 C19T31 1 4  4 Benning A30 G36 C19 T37 A41 G28 C19 T31 3 6  6 (Cultured) A30G36 C20 T36 A40 G29 C19 T31 1 11  11 A30 G36 C20 T36 A40 G29 C19 T31 113   94** A30 G36 C20 T36 A41 G28 C19 T31 1 44/61 or 82 82 A30 G36 C20T36 A41 G28 C19 T31 or 9 1 5 or 58 58 A30 G36 C20 T36 A40 G29 C19 T31 178 or 89 89 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 ND Lackland 2003A30 G36 C20 T36 A40 G29 C19 T31 1 2 AFB A30 G36 C20 T36 A40 G29 C19 T311 81 or 90 (Throat A30 G36 C20 T36 A40 G29 C19 T31 1 78  Swabs) A30 G36C20 T36 A41 G28 C19 T31   3*** No detection No detection No detection 73 ND MCRD San 2002 A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND Diego A30 G36C20 T36 A40 G29 C19 T31 1 3 ND (Throat A30 G36 C20 T36 No detection 1 3ND Swabs) No detection A40 G29 C19 T31 2 3 ND A30 G36 C20 T36 A40 G29C19 T31 3 No detection ND No detection No detection

TABLE 9C Base Composition Analysis of Bioagent Identifying Amplicons ofGroup A Streptococcus samples from Six Military Installations Obtainedwith Primer Pair Nos. 438 and 441 emm-type by gki gtr # of Mass emm-GeneLocation (Primer Pair ((Primer Pair Instances Spectrometry Sequencing(sample) Year No. 442) No. 443) 48  3  3 MCRD San 2002 A32 G35 C17 T32A39 G28 C16 T32 2 6  6 Diego A31 G35 C17 T33 A39 G28 C15 T33 1 28  28(Cultured) A30 G36 C17 T33 A39 G28 C16 T32 15  3 ND A32 G35 C17 T32 A39G28 C16 T32 6 3  3 NHRC San 2003 A32 G35 C17 T32 A39 G28 C16 T32 3 5, 58 5 Diego- A30 G36 C20 T30 A39 G28 C15 T33 6 6  6 Archive A31 G35 C17 T33A39 G28 C15 T33 1 11  11 (Cultured) A30 G36 C20 T30 A39 G28 C16 T32 312  12 A31 G35 C17 T33 A39 G28 C15 T33 1 22  22 A31 G35 C17 T33 A38 G29C15 T33 3 25, 75 75 A30 G36 C17 T33 A39 G28 C15 T33 4 44/61, 82, 9 44/61A30 G36 C18 T32 A39 G28 C15 T33 2 53, 91 91 A32 G35 C17 T32 A39 G28 C16T32 1 2  2 Ft. 2003 A30 G36 C17 T33 A39 G28 C15 T33 2 3  3 Leonard A32G35 C17 T32 A39 G28 C16 T32 1 4  4 Wood A31 G35 C17 T33 A39 G28 C15 T331 6  6 (Cultured) A31 G35 C17 T33 A39 G28 C15 T33 11  25 or 75 75 A30G36 C17 T33 A39 G28 C15 T33 1 25, 75, 33, 75 A30 G36 C17 T33 A39 G28 C15T33 34, 4, 52, 84 1 44/61 or 82 44/61 A30 G36 C18 T32 A39 G28 C15 T33 or9 2 5 or 58  5 A30 G36 C20 T30 A39 G28 C15 T33 3 1  1 Ft. Sill 2003 A30G36 C18 T32 A39 G28 C15 T33 2 3  3 (Cultured) A32 G35 C17 T32 A39 G28C16 T32 1 4  4 A31 G35 C17 T33 A39 G28 C15 T33 1 28  28 A30 G36 C17 T33A39 G28 C16 T32 1 3  3 Ft. 2003 A32 G35 C17 T32 A39 G28 C16 T32 1 4  4Benning A31 G35 C17 T33 A39 G28 C15 T33 3 6  6 (Cultured) A31 G35 C17T33 A39 G28 C15 T33 1 11  11 A30 G36 C20 T30 A39 G28 C16 T32 1 13   94**A30 G36 C19 T31 A39 G28 C15 T33 1 44/61 or 82 82 A30 G36 C18 T32 A39 G28C15 T33 or 9 1 5 or 58 58 A30 G36 C20 T30 A39 G28 C15 T33 1 78 or 89 89A30 G36 C18 T32 A39 G28 C15 T33 2 5 or 58 ND Lackland 2003 A30 G36 C20T30 A39 G28 C15 T33 1 2 AFB A30 G36 C17 T33 A39 G28 C15 T33 1 81 or 90(Throat A30 G36 C17 T33 A39 G28 C15 T33 1 78  Swabs) A30 G36 C18 T32 A39G28 C15 T33   3*** No detection No detection No detection 7 3 ND MCRDSan 2002 A32 G35 C17 T32 A39 G28 C16 T32 1 3 ND Diego No detection Nodetection 1 3 ND (Throat A32 G35 C17 T32 A39 G28 C16 T32 1 3 ND Swabs)A32 G35 C17 T32 No detection 2 3 ND A32 G35 C17 T32 No detection 3 Nodetection ND No detection No detection

Example 8 Design of Calibrant Polynucleotides Based on BioagentIdentifying Amplicons for Identification of Species of Bacteria(Bacterial Bioagent Identifying Amplicons)

This example describes the design of 19 calibrant polynucleotides basedon bacterial bioagent identifying amplicons corresponding to the primersof the broad surveillance set (Table 5) and the Bacillus anthracisdrill-down set (Table 6).

Calibration sequences were designed to simulate bacterial bioagentidentifying amplicons produced by the T modified primer pairs shown inTables 5 and 6 (primer names have the designation “TMOD”). Thecalibration sequences were chosen as a representative member of thesection of bacterial genome from specific bacterial species which wouldbe amplified by a given primer pair. The model bacterial species uponwhich the calibration sequences are based are also shown in Table 10.For example, the calibration sequence chosen to correspond to anamplicon produced by primer pair no. 361 is SEQ ID NO: 1445. In Table10, the forward (_F) or reverse (_R) primer name indicates thecoordinates of an extraction representing a gene of a standard referencebacterial genome to which the primer hybridizes e.g.: the forward primername 16S_EC_(—)713_(—)732_TMOD_F indicates that the forward primerhybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA inan E. coli reference sequence (in this case, the reference sequence isan extraction consisting of residues 4033120-4034661 of the genomicsequence of E. coli K12 (GenBank gi number 16127994). Additional genecoordinate reference information is shown in Table 11. The designation“TMOD” in the primer names indicates that the 5′ end of the primer hasbeen modified with a non-matched template T residue which prevents thePCR polymerase from adding non-templated adenosine residues to the 5′end of the amplification product, an occurrence which may result inmiscalculation of base composition from molecular mass data (videsupra).

The 19 calibration sequences described in Tables 10 and 11 were combinedinto a single calibration polynucleotide sequence (SEQ ID NO: 1464—whichis herein designated a “combination calibration polynucleotide”) whichwas then cloned into a pCR®—Blunt vector (Invitrogen, Carlsbad, Calif.).This combination calibration polynucleotide can be used in conjunctionwith the primers of Tables 5 or 6 as an internal standard to producecalibration amplicons for use in determination of the quantity of anybacterial bioagent. Thus, for example, when the combination calibrationpolynucleotide vector is present in an amplification reaction mixture, acalibration amplicon based on primer pair 346 (16S rRNA) will beproduced in an amplification reaction with primer pair 346 and acalibration amplicon based on primer pair 363 (rpoC) will be producedwith primer pair 363. Coordinates of each of the 19 calibrationsequences within the calibration polynucleotide (SEQ ID NO: 1464) areindicated in Table 11.

TABLE 10 Bacterial Primer Pairs for Production of Bacterial BioagentIdentifying Amplicons and Corresponding Representative CalibrationSequences Forward Reverse Calibration Primer Primer Calibration SequencePrimer (SEQ ID (SEQ ID Sequence Model (SEQ ID Pair No. Forward PrimerName NO:) Reverse Primer Name NO:) Species NO:) 36116S_EC_1090_1111_2_TMOD_F 697 16S_EC_1175_1196_TMOD_R 1398 Bacillus 1445anthracis 346 16S_EC_713_732_TMOD_F 202 16S_EC_789_809_TMOD_R 1110Bacillus 1446 anthracis 347 16S_EC_785_806_TMOD_F 56016S_EC_880_897_TMOD_R 1278 Bacillus 1447 anthracis 34816S_EC_960_981_TMOD_F 706 16S_EC_1054_1073_TMOD_R 895 Bacillus 1448anthracis 349 23S_EC_1826_1843_TMOD_F 401 23S_EC_1906_1924_TMOD_R 1156Bacillus 1449 anthracis 360 23S_EC_2646_2667_TMOD_F 40923S_EC_2745_2765_TMOD_R 1434 Bacillus 1450 anthracis 350CAPC_BA_274_303_TMOD_F 476 CAPC_BA_349_376_TMOD_R 1314 Bacillus 1451anthracis 351 CYA_BA_1353_1379_TMOD_F 355 CYA_BA_1448_1467_TMOD_R 1423Bacillus 1452 anthracis 352 INFB_EC_1365_1393_TMOD_F 687INFB_EC_1439_1467_TMOD_R 1411 Bacillus 1453 anthracis 353LEF_BA_756_781_TMOD_F 220 LEF_BA_843_872_TMOD_R 1394 Bacillus 1454anthracis 356 RPLB_EC_650_679_TMOD_F 449 RPLB_EC_739_762_TMOD_R 1380Clostridium 1455 botulinum 449 RPLB_EC_690_710_F 309 RPLB_EC_737_758_R1336 Clostridium 1456 botulinum 359 RPOB_EC_1845_1866_TMOD_F 659RPOB_EC_1909_1929_TMOD_R 1250 Yersinia 1457 Pestis 362RPOB_EC_3799_3821_TMOD_F 581 RPOB_EC_3862_3888_TMOD_R 1325 Burkholderia1458 mallei 363 RPOC_EC_2146_2174_TMOD_F 284 RPOC_EC_2227_2245_TMOD_R898 Burkholderia 1459 mallei 354 RPOC_EC_2218_2241_TMOD_F 405RPOC_EC_2313_2337_TMOD_R 1072 Bacillus 1460 anthracis 355SSPE_BA_115_137_TMOD_F 255 SSPE_BA_197_222_TMOD_R 1402 Bacillus 1461anthracis 367 TUFB_EC_957_979_TMOD_F 308 TUFB_EC_1034_1058_TMOD_R 1276Burkholderia 1462 mallei 358 VALS_EC_1105_1124_TMOD_F 385VALS_EC_1195_1218_TMOD_R 1093 Yersinia 1463 Pestis

TABLE 11 Primer Pair Gene Coordinate References and CalibrationPolynucleotide Sequence Coordinates within the Combination CalibrationPolynucleotide Coordinates of Gene Extraction Calibration Sequence inBacterial Coordinates Reference GenBank GI Combination Calibration Geneand of Genomic or Plasmid No. of Genomic (G) or Primer Polynucleotide(SEQ ID Species Sequence Plasmid (P) Sequence Pair No. NO: 1464) 16S E.coli 4033120 . . . 4034661 16127994 (G) 346  16 . . . 109 16S E. coli4033120 . . . 4034661 16127994 (G) 347  83 . . . 190 16S E. coli 4033120. . . 4034661 16127994 (G) 348 246 . . . 353 16S E. coli 4033120 . . .4034661 16127994 (G) 361 368 . . . 469 23S E. coli 4166220 . . . 416912316127994 (G) 349 743 . . . 837 23S E. coli 4166220 . . . 416912316127994 (G) 360 865 . . . 981 rpoB E. coli. 4178823 . . . 418285116127994 (G) 359 1591 . . . 1672 (complement strand) rpoB E. coli4178823 . . . 4182851 16127994 (G) 362 2081 . . . 2167 (complementstrand) rpoC E. coli 4182928 . . . 4187151 16127994 (G) 354 1810 . . .1926 rpoC E. coli 4182928 . . . 4187151 16127994 (G) 363 2183 . . . 2279infB E. coli 3313655 . . . 3310983 16127994 (G) 352 1692 . . . 1791(complement strand) tufB E. coli 4173523 . . . 4174707 16127994 (G) 3672400 . . . 2498 rplB E. coli 3449001 . . . 3448180 16127994 (G) 356 1945. . . 2060 rplB E. coli 3449001 . . . 3448180 16127994 (G) 449 1986 . .. 2055 valS E. coli 4481405 . . . 4478550 16127994 (G) 358 1462 . . .1572 (complement strand) capC 56074 . . . 55628  6470151 (P) 350 2517 .. . 2616 B. anthracis (complement strand) cya 156626 . . . 154288 4894216 (P) 351 1338 . . . 1449 B. anthracis (complement strand) lef127442 . . . 129921  4894216 (P) 353 1121 . . . 1234 B. anthracis sspE226496 . . . 226783 30253828 (G) 355 1007-1104 B. anthracis

Example 9 Use of a Calibration Polynucleotide for Determining theQuantity of Bacillus Anthracis in a Sample Containing a Mixture ofMicrobes

The process described in this example is shown in FIG. 2. The capC geneis a gene involved in capsule synthesis which resides on the pX02plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 10 and11) was designed to identify Bacillus anthracis via production of abacterial bioagent identifying amplicon. Known quantities of thecombination calibration polynucleotide vector described in Example 8were added to amplification mixtures containing bacterial bioagentnucleic acid from a mixture of microbes which included the Ames strainof Bacillus anthracis. Upon amplification of the bacterial bioagentnucleic acid and the combination calibration polynucleotide vector withprimer pair no. 350, bacterial bioagent identifying amplicons andcalibration amplicons were obtained and characterized by massspectrometry. A mass spectrum measured for the amplification reaction isshown in FIG. 7. The molecular masses of the bioagent identifyingamplicons provided the means for identification of the bioagent fromwhich they were obtained (Ames strain of Bacillus anthracis) and themolecular masses of the calibration amplicons provided the means fortheir identification as well. The relationship between the abundance(peak height) of the calibration amplicon signals and the bacterialbioagent identifying amplicon signals provides the means of calculationof the copies of the pX02 plasmid of the Ames strain of Bacillusanthracis. Methods of calculating quantities of molecules based oninternal calibration procedures are well known to those of ordinaryskill in the art.

Averaging the results of 10 repetitions of the experiment describedabove, enabled a calculation that indicated that the quantity of Amesstrain of Bacillus anthracis present in the sample corresponds toapproximately 10 copies of pX02 plasmid.

Example 10 Triangulation Genotyping Analysis of Campylobacter Species

A series of triangulation genotyping analysis primers were designed asdescribed in Example 1 with the objective of identification of differentstrains of Campylobacter jejuni. The primers are listed in Table 12 withthe designation “CJST_CJ.” Housekeeping genes to which the primershybridize and produce bioagent identifying amplicons include: tkt(transketolase), glyA (serine hydroxymethyltransferase), gltA (citratesynthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase),pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).

TABLE 12 Campylobacter Genotyping Primer Pairs Primer Pair ForwardPrimer Reverse Primer No. Forward Primer Name (SEQ ID NO:) ReversePrimer Name (SEQ ID NO:) Target Gene 1053 CJST_CJ_1080_1110_F 681CJST_CJ_1166_1198_R 1022 gltA 1047 CJST_CJ_584_616_F 315CJST_CJ_663_692_R 1379 glnA 1048 CJST_CJ_360_394_F 346 CJST_CJ_442_476_R955 aspA 1049 CJST_CJ_2636_2668_F 504 CJST_CJ_2753_2777_R 1409 tkt 1054CJST_CJ_2060_2090_F 323 CJST_CJ_2148_2174_R 1068 pgm 1064CJST_CJ_1680_1713_F 479 CJST_CJ_1795_1822_R 938 glyA

The primers were used to amplify nucleic acid from 50 food productsamples provided by the USDA, 25 of which contained Campylobacter jejuniand 25 of which contained Campylobacter coli. Primers used in this studywere developed primarily for the discrimination of Campylobacter jejuniclonal complexes and for distinguishing Campylobacter jejuni fromCampylobacter coli. Finer discrimination between Campylobacter colitypes is also possible by using specific primers targeted to loci whereclosely-related Campylobacter coli isolates demonstrate polymorphismsbetween strains. The conclusions of the comparison of base compositionanalysis with sequence analysis are shown in Tables 1 3A-C.

TABLE 13A Results of Base Composition Analysis of 50 CampylobacterSamples with Drill-down MLST Primer Pair Nos: 1048 and 1047 Base BaseComposition of Composition of MLST type or Bioagent Bioagent Clonal MLSTType Identifying Identifying Complex by or Clonal Amplicon Amplicon BaseComplex by Obtained with Obtained with Isolate Composition SequencePrimer Pair No: Primer Pair Group Species origin analysis analysisStrain 1048 (aspA) No: 1047 (glnA) J-1 C. jejuni Goose ST 690/ ST 991RM3673 A30 G25 C16 T46 A47 G21 C16 T25 692/707/991 J-2 C. jejuni HumanComplex ST 356, RM4192 A30 G25 C16 T46 A48 G21 C17 T23 206/48/353complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A30 G25 C15 T47A48 G21 C18 T22 354/179 J-4 C. jejuni Human Complex 257 ST 257, RM4197A30 G25 C16 T46 A48 G21 C18 T22 complex 257 J-5 C. jejuni Human Complex52 ST 52, RM4277 A30 G25 C16 T46 A48 G21 C17 T23 complex 52 J-6 C.jejuni Human Complex 443 ST 51, RM4275 A30 G25 C15 T47 A48 G21 C17 T23complex RM4279 A30 G25 C15 T47 A48 G21 C17 T23 443 J-7 C. jejuni HumanComplex 42 ST 604, RM1864 A30 G25 C15 T47 A48 G21 C18 T22 complex 42 J-8C. jejuni Human Complex ST 362, RM3193 A30 G25 C15 T47 A48 G21 C18 T2242/49/362 complex 362 J-9 C. jejuni Human Complex ST 147, RM3203 A30 G25C15 T47 A47 G21 C18 T23 45/283 Complex 45 C. jejuni Human Consistent ST828 RM4183 A31 G27 C20 T39 A48 G21 C16 T24 C-1 C. coli with 74 ST 832RM1169 A31 G27 C20 T39 A48 G21 C16 T24 closely ST 1056 RM1857 A31 G27C20 T39 A48 G21 C16 T24 Poultry related ST 889 RM1166 A31 G27 C20 T39A48 G21 C16 T24 sequence ST 829 RM1182 A31 G27 C20 T39 A48 G21 C16 T24types (none ST 1050 RM1518 A31 G27 C20 T39 A48 G21 C16 T24 belong to aST 1051 RM1521 A31 G27 C20 T39 A48 G21 C16 T24 clonal ST 1053 RM1523 A31G27 C20 T39 A48 G21 C16 T24 complex) ST 1055 RM1527 A31 G27 C20 T39 A48G21 C16 T24 ST 1017 RM1529 A31 G27 C20 T39 A48 G21 C16 T24 ST 860 RM1840A31 G27 C20 T39 A48 G21 C16 T24 ST 1063 RM2219 A31 G27 C20 T39 A48 G21C16 T24 ST 1066 RM2241 A31 G27 C20 T39 A48 G21 C16 T24 ST 1067 RM2243A31 G27 C20 T39 A48 G21 C16 T24 ST 1068 RM2439 A31 G27 C20 T39 A48 G21C16 T24 Swine ST 1016 RM3230 A31 G27 C20 T39 A48 G21 C16 T24 ST 1069RM3231 A31 G27 C20 T39 A48 G21 C16 T24 ST 1061 RM1904 A31 G27 C20 T39A48 G21 C16 T24 Unknown ST 825 RM1534 A31 G27 C20 T39 A48 G21 C16 T24 ST901 RM1505 A31 G27 C20 T39 A48 G21 C16 T24 C-2 C. coli Human ST 895 ST895 RM1532 A31 G27 C19 T40 A48 G21 C16 T24 C-3 C. coli PoultryConsistent ST 1064 RM2223 A31 G27 C20 T39 A48 G21 C16 T24 with 63 ST1082 RM1178 A31 G27 C20 T39 A48 G21 C16 T24 closely ST 1054 RM1525 A31G27 C20 T39 A48 G21 C16 T24 related ST 1049 RM1517 A31 G27 C20 T39 A48G21 C16 T24 Marmoset sequence ST 891 RM1531 A31 G27 C20 T39 A48 G21 C16T24 types (none belong to a clonal complex)

TABLE 13B Results of Base Composition Analysis of 50 CampylobacterSamples with Drill- down MLST Primer Pair Nos: 1053 and 1064 Base BaseComposition of Composition of MLST type or Bioagent Bioagent Clonal MLSTType Identifying Identifying Complex by or Clonal Amplicon Amplicon BaseComplex by Obtained with Obtained with Isolate Composition SequencePrimer Pair Primer Pair Group Species origin analysis analysis StrainNo: 1053 (gltA) No: 1064 (glyA) J-1 C. jejuni Goose ST 690/ ST 991RM3673 A24 G25 C23 T47 A40 G29 C29 T45 692/707/991 J-2 C. jejuni HumanComplex ST 356, RM4192 A24 G25 C23 T47 A40 G29 C29 T45 206/48/353complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A24 G25 C23 T47A40 G29 C29 T45 354/179 J-4 C. jejuni Human Complex 257 ST 257, RM4197A24 G25 C23 T47 A40 G29 C29 T45 complex 257 J-5 C. jejuni Human Complex52 ST 52, RM4277 A24 G25 C23 T47 A39 G30 C26 T48 complex 52 J-6 C.jejuni Human Complex 443 ST 51, RM4275 A24 G25 C23 T47 A39 G30 C28 T46complex RM4279 A24 G25 C23 T47 A39 G30 C28 T46 443 J-7 C. jejuni HumanComplex 42 ST 604, RM1864 A24 G25 C23 T47 A39 G30 C26 T48 complex 42 J-8C. jejuni Human Complex ST 362, RM3193 A24 G25 C23 T47 A38 G31 C28 T4642/49/362 complex 362 J-9 C. jejuni Human Complex ST 147, RM3203 A24 G25C23 T47 A38 G31 C28 T46 45/283 Complex 45 C. jejuni Human Consistent ST828 RM4183 A23 G24 C26 T46 A39 G30 C27 T47 C-1 C. coli with 74 ST 832RM1169 A23 G24 C26 T46 A39 G30 C27 T47 closely ST 1056 RM1857 A23 G24C26 T46 A39 G30 C27 T47 Poultry related ST 889 RM1166 A23 G24 C26 T46A39 G30 C27 T47 sequence ST 829 RM1182 A23 G24 C26 T46 A39 G30 C27 T47types (none ST 1050 RM1518 A23 G24 C26 T46 A39 G30 C27 T47 belong to aST 1051 RM1521 A23 G24 C26 T46 A39 G30 C27 T47 clonal ST 1053 RM1523 A23G24 C26 T46 A39 G30 C27 T47 complex) ST 1055 RM1527 A23 G24 C26 T46 A39G30 C27 T47 ST 1017 RM1529 A23 G24 C26 T46 A39 G30 C27 T47 ST 860 RM1840A23 G24 C26 T46 A39 G30 C27 T47 ST 1063 RM2219 A23 G24 C26 T46 A39 G30C27 T47 ST 1066 RM2241 A23 G24 C26 T46 A39 G30 C27 T47 ST 1067 RM2243A23 G24 C26 T46 A39 G30 C27 T47 ST 1068 RM2439 A23 G24 C26 T46 A39 G30C27 T47 Swine ST 1016 RM3230 A23 G24 C26 T46 A39 G30 C27 T47 ST 1069RM3231 A23 G24 C26 T46 NO DATA ST 1061 RM1904 A23 G24 C26 T46 A39 G30C27 T47 Unknown ST 825 RM1534 A23 G24 C26 T46 A39 G30 C27 T47 ST 901RM1505 A23 G24 C26 T46 A39 G30 C27 T47 C-2 C. coli Human ST 895 ST 895RM1532 A23 G24 C26 T46 A39 G30 C27 T47 C-3 C. coli Poultry Consistent ST1064 RM2223 A23 G24 C26 T46 A39 G30 C27 T47 with 63 ST 1082 RM1178 A23G24 C26 T46 A39 G30 C27 T47 closely ST 1054 RM1525 A23 G24 C25 T47 A39G30 C27 T47 related ST 1049 RM1517 A23 G24 C26 T46 A39 G30 C27 T47Marmoset sequence ST 891 RM1531 A23 G24 C26 T46 A39 G30 C27 T47 types(none belong to a clonal complex)

TABLE 13C Results of Base Composition Analysis of 50 CampylobacterSamples with Drill- down MLST Primer Pair Nos: 1054 and 1049 Base BaseComposition of Composition of MLST type or Bioagent Bioagent Clonal MLSTType Identifying Identifying Complex by or Clonal Amplicon Amplicon BaseComplex by Obtained with Obtained with Isolate Composition SequencePrimer Pair No: Primer Pair Group Species origin analysis analysisStrain 1054 (pgm) No: 1049 (tkt) J-1 C. jejuni Goose ST 690/ ST 991RM3673 A26 G33 C18 T38 A41 G28 C35 T38 692/707/991 J-2 C. jejuni HumanComplex ST 356, RM4192 A26 G33 C19 T37 A41 G28 C36 T37 206/48/353complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A27 G32 C19 T37A42 G28 C36 T36 354/179 J-4 C. jejuni Human Complex 257 ST 257, RM4197A27 G32 C19 T37 A41 G29 C35 T37 complex 257 J-5 C. jejuni Human complex52 ST 52, RM4277 A26 G33 C18 T38 A41 G28 C36 T37 complex 52 J-6 C.jejuni Human Complex 443 ST 51, RM4275 A27 G31 C19 T38 A41 G28 C36 T37complex RM4279 A27 G31 C19 T38 A41 G28 C36 T37 443 J-7 C. jejuni HumanComplex 42 ST 604, RM1864 A27 G32 C19 T37 A42 G28 C35 T37 complex 42 J-8C. jejuni Human Complex ST 362, RM3193 A26 G33 C19 T37 A42 G28 C35 T3742/49/362 complex 362 J-9 C. jejuni Human Complex ST 147, RM3203 A28 G31C19 T37 A43 G28 C36 T35 45/283 Complex 45 C. jejuni Human Consistent ST828 RM4183 A27 G30 C19 T39 A46 G28 C32 T36 C-1 C. coli with 74 ST 832RM1169 A27 G30 C19 T39 A46 G28 C32 T36 closely ST 1056 RM1857 A27 G30C19 T39 A46 G28 C32 T36 Poultry related ST 889 RM1166 A27 G30 C19 T39A46 G28 C32 T36 sequence ST 829 RM1182 A27 G30 C19 T39 A46 G28 C32 T36types (none ST 1050 RM1518 A27 G30 C19 T39 A46 G28 C32 T36 belong to aST 1051 RM1521 A27 G30 C19 T39 A46 G28 C32 T36 clonal ST 1053 RM1523 A27G30 C19 T39 A46 G28 C32 T36 complex) ST 1055 RM1527 A27 G30 C19 T39 A46G28 C32 T36 ST 1017 RM1529 A27 G30 C19 T39 A46 G28 C32 T36 ST 860 RM1840A27 G30 C19 T39 A46 G28 C32 T36 ST 1063 RM2219 A27 G30 C19 T39 A46 G28C32 T36 ST 1066 RM2241 A27 G30 C19 T39 A46 G28 C32 T36 ST 1067 RM2243A27 G30 C19 T39 A46 G28 C32 T36 ST 1068 RM2439 A27 G30 C19 T39 A46 G28C32 T36 Swine ST 1016 RM3230 A27 G30 C19 T39 A39 G30 C27 T47 ST 1069RM3231 A27 G30 C19 T39 A46 G28 C32 T36 ST 1061 RM1904 A27 G30 C19 T39A46 G28 C32 T36 Unknown ST 825 RM1534 A27 G30 C19 T39 A46 G28 C32 T36 ST901 RM1505 A27 G30 C19 T39 A46 G28 C32 T36 C-2 C. coli Human ST 895 ST895 RM1532 A27 G30 C19 T39 A46 G28 C32 T36 C-3 C. coli PoultryConsistent ST 1064 RM2223 A27 G30 C19 T39 A46 G28 C32 T36 with 63 ST1082 RM1178 A27 G30 C19 T39 A46 G28 C32 T36 closely ST 1054 RM1525 A27G30 C19 T39 A46 G28 C32 T36 related ST 1049 RM1517 A27 G30 C19 T39 A46G28 C32 T36 Marmoset sequence ST 891 RM1531 A27 G30 C19 T39 A46 G28 C32T36 types (none belong to a clonal complex)

The base composition analysis method was successful in identification of12 different strain groups. Campylobacter jejuni and Campylobacter coliare generally differentiated by all loci. Ten clearly differentiatedCampylobacter jejuni isolates and 2 major Campylobacter coli groups wereidentified even though the primers were designed for strain typing ofCampylobacter jejuni. One isolate (RM4183) which was designated asCampylobacter jejuni was found to group with Campylobacter coli and alsoappears to actually be Campylobacter coli by full MLST sequencing.

Example 11 Identification of Acinetobacter baumannii Using Broad RangeSurvey and Division-Wide Primers in Epidemiological Surveillance

To test the capability of the broad range survey and division-wideprimer sets of Table 5 in identification of Acinetobacter species, 183clinical samples were obtained from individuals participating in, or incontact with individuals participating in Operation Iraqi Freedom(including US service personnel, US civilian patients at the Walter ReedArmy Institute of Research (WRAIR), medical staff, Iraqi civilians andenemy prisoners. In addition, 34 environmental samples were obtainedfrom hospitals in Iraq, Kuwait, Germany, the United States and the USNSComfort, a hospital ship.

Upon amplification of nucleic acid obtained from the clinical samples,primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 5) all producedbacterial bioagent amplicons which identified Acinetobacter baumannii in215 of 217 samples. The organism Klebsiella pneumoniae was identified inthe remaining two samples. In addition, 14 different strain types(containing single nucleotide polymorphisms relative to a referencestrain of Acinetobacter baumannii) were identified and assignedarbitrary numbers from 1 to 14. Strain type 1 was found in 134 of thesample isolates and strains 3 and 7 were found in 46 and 9 of theisolates respectively.

The epidemiology of strain type 7 of Acinetobacter baumannii wasinvestigated. Strain 7 was found in 4 patients and 5 environmentalsamples (from field hospitals in Iraq and Kuwait). The index patientinfected with strain 7 was a pre-war patient who had a traumaticamputation in March of 2003 and was treated at a Kuwaiti hospital. Thepatient was subsequently transferred to a hospital in Germany and thento WRAIR. Two other patients from Kuwait infected with strain 7 werefound to be non-infectious and were not further monitored. The fourthpatient was diagnosed with a strain 7 infection in September of 2003 atWRAIR. Since the fourth patient was not related involved in OperationIraqi Freedom, it was inferred that the fourth patient was the subjectof a nosocomial infection acquired at WRAIk as a result of the spread ofstrain 7 from the index patient.

The epidemiology of strain type 3 of Acinetobacter baumannii was alsoinvestigated. Strain type 3 was found in 46 samples, all of which werefrom patients (US service members, Iraqi civilians and enemy prisoners)who were treated on the USNS Comfort hospital ship and subsequentlyreturned to Iraq or Kuwait. The occurrence of strain type 3 in a singlelocale may provide evidence that at least some of the infections at thatlocale were a result of nosocomial infections.

This example thus illustrates an embodiment of the present inventionwherein the methods of analysis of bacterial bioagent identifyingamplicons provide the means for epidemiological surveillance.

Example 12 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Acinetobacter baumanii

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, anadditional 21 primer pairs were selected based on analysis ofhousekeeping genes of the genus Acinetobacter. Genes to which thedrill-down triangulation genotyping analysis primers hybridize forproduction of bacterial bioagent identifying amplicons includeanthranilate synthase component I (trpE), adenylate kinase (adk),adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphatephospho-hydratase (ppa). These 21 primer pairs are indicated withreference to sequence listings in Table 14. Primer pair numbers1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers1155-1157 hybridize to and amplify segments of adk. Primer pair numbers1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers1165-1170 hybridize to and amplify segments of fumC. Primer pair number1171 hybridizes to and amplifies a segment of ppa. Primer pair numbers:2846-2848 hybridize to and amplify segments of the parC gene of DNAtopoisomerase which include a codon known to confer quinolone drugresistance upon sub-types of Acinetobacter baumannii. Primer pairnumbers 2852-2854 hybridize to and amplify segments of the gyrA gene ofDNA gyrase which include a codon known to confer quinolone drugresistance upon sub-types of Acinetobacter baumannii. Primer pairnumbers 2922 and 2972 are speciating primers which are useful foridentifying different species members of the genus Acinetobacter. Theprimer names given in Table 14A (with the exception of primer pairnumbers 2846-2848, 2852-2854) indicate the coordinates to which theprimers hybridize to a reference sequence which comprises aconcatenation of the genes TrpE, efp (elongation factor p), adk, mutT,fumC, and ppa. For example, the forward primer of primer pair 1151 isnamed AB_MLST-11-OIF007_(—)62_(—)91_F because it hybridizes to theAcinetobacter primer reference sequence of strain type 11 in sample 007of Operation Iraqi Freedom (OIF) at positions 62 to 91. DNA wassequenced from strain type 11 and from this sequence data and anartificial concatenated sequence of partial gene extractions wasassembled for use in design of the triangulation genotyping analysisprimers. The stretches of arbitrary residues “N”s in the concatenatedsequence were added for the convenience of separation of the partialgene extractions (40N for AB_MLST (SEQ ID NO: 1444)).

The hybridization coordinates of primer pair numbers 2846-2848 are withrespect to GenBank Accession number X95819. The hybridizationcoordinates of primer pair numbers 2852-2854 are with respect to GenBankAccession number AY642140. Sequence residue “I” appearing in the forwardand reverse primers of primer pair number 2972 represents inosine.

TABLE 14A Triangulation Genotyping Analysis Primer Pairs forIdentification of Sub-species characteristics (Strain Type) of Membersof the Bacterial Genus Acinetobacter Primer Forward Primer ReversePrimer Pair No. Forward Primer Name (SEQ ID NO:) Reverse Primer Name(SEQ ID NO:) 1151 AB_MLST-11-OIF007_62_91_F 454AB_MLST-11-OIF007_169_203_R 1418 1152 AB_MLST-11-OIF007_185_214_F 243AB_MLST-11-OIF007_291_324_R 969 1153 AB_MLST-11-OIF007_260_289_F 541AB_MLST-11-OIF007_364_393_R 1400 1154 AB_MLST-11-OIF007_206_239_F 436AB_MLST-11-OIF007_318_344_R 1036 1155 AB_MLST-11-OIF007_522_552_F 378AB_MLST-11-OIF007_587_610_R 1392 1156 AB_MLST-11-OIF007_547_571_F 250AB_MLST-11-OIF007_656_686_R 902 1157 AB_MLST-11-OIF007_601_627_F 256AB_MLST-11-OIF007_710_736_R 881 1158 AB_MLST-11-OIF007_1202_1225_F 384AB_MLST-11-OIF007_1266_1296_R 878 1159 AB_MLST-11-OIF007_1202_1225_F 384AB_MLST-11-OIF007_1299_1316_R 1199 1160 AB_MLST-11-OIF007_1234_1264_F694 AB_MLST-11-OIF007_1335_1362_R 1215 1161AB_MLST-11-OIF007_1327_1356_F 225 AB_MLST-11-OIF007_1422_1448_R 12121162 AB_MLST-11-OIF007_1345_1369_F 383 AB_MLST-11-OIF007_1470_1494_R1083 1163 AB_MLST-11-OIF007_1351_1375_F 662AB_MLST-11-OIF007_1470_1494_R 1083 1164 AB_MLST-11-OIF007_1387_1412_F422 AB_MLST-11-OIF007_1470_1494_R 1083 1165AB_MLST-11-OIF007_1542_1569_F 194 AB_MLST-11-OIF007_1656_1680_R 11731166 AB_MLST-11-OIF007_1566_1593_F 684 AB_MLST-11-OIF007_1656_1680_R1173 1167 AB_MLST-11-OIF007_1611_1638_F 375AB_MLST-11-OIF007_1731_1757_R 890 1168 AB_MLST-11-OIF007_1726_1752_F 182AB_MLST-11-OIF007_1790_1821_R 1195 1169 AB_MLST-11-OIF007_1792_1826_F656 AB_MLST-11-OIF007_1876_1909_R 1151 1170AB_MLST-11-OIF007_1792_1826_F 656 AB_MLST-11-OIF007_1895_1927_R 12241171 AB_MLST-11-OIF007_1970_2002_F 618 AB_MLST-11-OIF007_2097_2118_R1157 2846 PARC_X95819_33_58_F 302 PARC_X95819_121_153_R 852 2847PARC_X95819_33_58_F 199 PARC_X95819_157_178_R 889 2848PARC_X95819_33_58_F 596 PARC_X95819_97_128_R 1169 2852GYRA_AY642140_−1_24_F 150 GYRA_AY642140_71_100_R 1242 2853GYRA_AY642140_26_54_F 166 GYRA_AY642140_121_146_R 1069 2854GYRA_AY642140_26_54_F 166 GYRA_AY642140_58_89_R 1168 2922AB_MLST-11-OIF007_991_1018_F 583 AB_MLST-11-OIF007_1110_1137_R 923 2972AB_MLST-11-OIF007_1007_1034_F 592 AB_MLST-11-OIF007_1126_1153_R 924

TABLE 14B Triangulation Genotyping Analysis Primer Pairs forIdentification of Sub-species characteristics (Strain Type) of Membersof the Bacterial Genus Acinetobacter Forward Reverse Primer PrimerPrimer Pair No. (SEQ ID NO:) SEQUENCE (SEQ ID NO: SEQUENCE 1151 454TGAGATTGCTGAACATTTAATGCTGATTGA 1418 TTGTACATTTGAAACAATATGCATGACATGTGAAT1152 243 TATTGTTTCAAATGTACAAGGTGAAGTGCG  969TCACAGGTTCACTTCATCAATAATTTCCATTGC 1153 541TGGAACGTTATCAGGTGCCCCAAAAATTCG 1400 TTGCAATCGACATATCCATTTCACCATGCC 1154436 TGAAGTGCGTGATGATATCGATGCACTTGATGTA 1036 TCCGCCAAAAACTCCCCTTTTCACAGG1155 378 TCGGTTTAGTAAAAGAACGTATTGCTCAACC 1392 TTCTGCTTGAGGAATAGTGCGTGG1156 250 TCAACCTGACTGCGTGAATGGTTGT  902 TACGTTCTACGATTTCTTCATCAGGTACATC1157 256 TCAAGCAGAAGCTTTGGAAGAAGAAGG  881 TACAACGTGATAAACACGACCAGAAGC1158 384 TCGTGCCCGCAATTTGCATAAAGC  878 TAATGCCGGGTAGTGCAATCCATTCTTCTAG1159 384 TCGTGCCCGCAATTTGCATAAAGC 1199 TGCACCTGCGGTCGAGCG 1160 694TTGTAGCACAGCAAGGCAAATTTCCTCAAAC 1215 TGCCATCCATAATCACGCCATACTGACG 1161225 TAGGTTTACGTCAGTATGGCGTGATTATGG 1212 TGCCAGTTTCCACATTTCACGTTCGTG 1162383 TCGTGATTATGGATGGCAACGTGAA 1083 TCGCTTGAGTGTACTGATGATTGCG 1163 662TTATGGATGGCAACGTGAAACGCGT 1083 TCGCTTGAGTGTAGTCATGATTGCG 1164 422TCTTTGCCATTGAAGATGACTTAAGC 1083 TCGCTTGAGTGTAGTCATGATTGCG 1165 194TACTAGCGGTAAGCTTAAACAAGATTGC 1173 TGAGTCGGGTTCACTTTACCTGGCA 1166 684TTGCCAATGATATTCGTTGGTTAGCAAG 1173 TGAGTCGGGTTCACTTTACCTGGCA 1167 375TCGGCGAAATCCGTATTCCTGAAAATGA  890 TACCGGAAGCACCAGCGACATTAATAG 1168 182TACCACTATTAATGTCGCTGGTGCTTC 1195 TGCAACTGAATAGATTGCAGTAAGTTATAAGC 1169656 TTATAACTTACTGCAATCTATTCAGTTGCTTGGT 1151TGAATTATGCAAGAAGTGATCAATTTTCTCACGA G 1170 656TTATAACTTACTGCAATCTATTCAGTTGCTTGGT 1224TGCCGTAACTAACATAAGAGAATTATGCAAGAA G 1171 618TGGTTATGTACCAAATACTTTGTCTGAAGATGG 1157 TGACGGCATCGATACCACCGTC 2846 302TCCAAAAAAATCAGCGCGTACAGTGG  852 TAAAGGATAGCGGTAACTAAATGGCTGAGCCAT 2847199 TACTTGGTAAATACCACCCACATGGTGA  889 TACCCCAGTTCCCCTGACCTTC 2848 596TGGTAAATACCACCCACATGGTGAC 1169 TGAGCCATGAGTACCATGGCTTCATAACATGC 2852 150TAAATCTGCCCGTGTCGTCGGTGAC 1242 TGCTAAAGTCTTGAGCCATACGAACAATGG 2853 166TAATCGGTAAATATCACCCGCATGGTGAC 1069 TCGATCGAACCGAAGTTACCCTGACC 2854 166TAATCGGTAAATATCACCCGCATGGTGAC 1168 TGAGCCATACGAACAATGGTTTCATAAACAGC 2922583 TGGGCGATGCTGCGAAATGGTTAAAAGA  923 TAGTATCACCACGTACACCCGCATCAGT 2972592 TGGGIGATGCTGCIAAATGGTTAAAAGA  924 TAGTATCACCACGTACICCIGGATCAGT

Analysis of bioagent identifying amplicons obtained using the primers ofTable 14B for over 200 samples from Operation Iraqi Freedom resulted inthe identification of 50 distinct strain type clusters. The largestcluster, designated strain type 11 (ST11) includes 42 sample isolates,all of which were obtained from US service personnel and Iraqi civilianstreated at the 28^(th) Combat Support Hospital in Baghdad. Several ofthese individuals were also treated on the hospital ship USNS Comfort.These observations are indicative of significant epidemiologicalcorrelation/linkage.

All of the sample isolates were tested against a broad panel ofantibiotics to characterize their antibodies resistance profiles. As anexample of a representative result from antibiotic susceptibilitytesting, ST11 was found to consist of four different clusters ofisolates, each with a varying degree of sensitivity/resistance to thevarious antibiotics tested which included penicillins, extended spectrumpenicillins, cephalosporins, carbepenem, protein synthesis inhibitors,nucleic acid synthesis inhibitors, anti-metabolites, and anti-cellmembrane antibiotics. Thus, the genotyping power of bacterial bioagentidentifying amplicons, particularly drill-down bacterial bioagentidentifying amplicons, has the potential to increase the understandingof the transmission of infections in combat casualties, to identify thesource of infection in the environment, to track hospital transmissionof nosocomial infections, and to rapidly characterize drug-resistanceprofiles which enable development of effective infection controlmeasures on a time-scale previously not achievable.

Example 13 Triangulation Genotyping Analysis and Codon Analysis ofAcinetobacter baumannii Samples from Two Health Care Facilities

In this investigation, 88 clinical samples were obtained from WalterReed Hospital and 95 clinical samples were obtained from NorthwesternMedical Center. All samples from both healthcare facilities weresuspected of containing sub-types of Acinetobacter baumannii, at leastsome of which were expected to be resistant to quinolone drugs. Each ofthe 183 samples was analyzed by the method of the present invention. DNAwas extracted from each of the samples and amplified with eighttriangulation genotyping analysis primer pairs represented by primerpair numbers: 1151, 1156, 1158, 1160, 1165, 1167, 1170, and 1171. TheDNA was also amplified with speciating primer pair number 2922 and codonanalysis primer pair numbers 2846-2848 which interrogate a codon presentin the parC gene, and primer pair numbers 2852-2854 which bracket acodon present in the gyrA gene. The parC and gyrA codon mutations areboth responsible for causing drug resistance in Acinetobacter baumannii.During evolution of drug resistant strains, the gyrA mutation usuallyoccurs before the parC mutation. Amplification products were measured byESI-TOF mass spectrometry as indicated in Example 4. The basecompositions of the amplification products were calculated from theaverage molecular masses of the amplification products and are shown inTables 15-18. The entries in each of the tables are grouped according tostrain type number, which is an arbitrary number assigned toAcinetobacter baumannii strains in the order of observance beginningfrom the triangulation genotyping analysis OIF genotyping studydescribed in Example 12. For example, strain type 11 which appears insamples from the Walter Reed Hospital is the same strain as the straintype 11 mentioned in Example 12. Ibis# refers to the order in which eachsample was analyzed. Isolate refers to the original sample isolatenumbering system used at the location from which the samples wereobtained (either Walter Reed Hospital or Northwestern Medical Center).ST=strain type. ND=not detected. Base compositions highlighted with boldtype indicate that the base composition is a unique base composition forthe amplification product obtained with the pair of primers indicated.

TABLE 15A Base Compositions of Amplification Products of 88 A. baumanniiSamples Obtained from Walter Reed Hospital and Amplified with CodonAnalysis Primer Pairs Targeting the gyrA Gene PP No: 2852 PP No: 2853 PPNo: 2854 Species Ibis# Isolate ST gyrA gyrA gyrA A. baumannii 20 1082 1A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 13  854 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 22 1162 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 27 1230 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 31 1367 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 37 1459 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 55 1700 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 64 1777 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 73 1861 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 74 1877 10 NDA29G28C21T43 A17G13C13T21 A. baumannii 86 1972 10 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  3  684 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  6  720 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  7  726 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 19 1079 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 21 1123 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 23 1188 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 33 1417 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 34 1431 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 38 1496 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 40 1523 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 42 1640 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 50 1666 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 51 1668 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 52 1695 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 65 1781 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 44 1649 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii   49A   1658.1 12 A25G23C22T31A29G28C21T43 A17G13C13T21 A. baumannii   49B   1658.2 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 56 1707 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 80 1893 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  5  693 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  8  749 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 10  839 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 14  865 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 16  888 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 29 1326 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 35 1440 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 41 1524 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 46 1652 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 47 1653 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 48 1657 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 57 1709 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 61 1727 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 63 1762 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 67 1806 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 75 1881 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 77 1886 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  1  649 46 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  2  653 46 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 39 1497 16 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 24 1198 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 28 1243 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 43 1648 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 62 1746 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  4  689 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 68 1822 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 69   1823A 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 70   1823B 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 71 1826 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 72 1860 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 81 1924 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 82 1929 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 85 1966 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 11  841 3 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 32 1415 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 45 1651 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 54 1697 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 58 1712 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 60 1725 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 66 1802 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 76 1883 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 78 1891 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 79 1892 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 83 1947 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 84 1964 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 53 1696 24 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 36 1458 49 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 59 1716 9 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii  9  805 30 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 18  967 39 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 30 1322 48 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 26 1218 50 A25G23C22T31A29G28C22T42 A17G13C14T20 A. sp. 13TU 15  875 A1 A25G23C22T31A29G28C22T42 A17G13C14T20 A. sp. 13TU 17  895 A1 A25G23C22T31A29G28C22T42 A17G13C14T20 A. sp. 3 12  853 B7 A25G22C22T32 A30G29C22T40A17G13C14T20 A. johnsonii 25 1202 NEW1 A25G22C22T32 A30G29C22T40A17G13C14T20 A. sp. 2082 87 2082 NEW2 A25G22C22T32 A31G28C22T40A17G13C14T20

TABLE 15B Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with Codon AnalysisPrimer Pairs Targeting the parC Gene PP No: 2846 PP No: 2847 PP No: 2848Species Ibis# Isolate ST parC parC parC A. baumannii 20 1082 1A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 13  854 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 22 1162 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 27 1230 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 31 1367 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 37 1459 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 55 1700 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 64 1777 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 73 1861 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 74 1877 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 86 1972 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  3  684 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  6  720 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  7  726 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 19 1079 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 21 1123 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 23 1188 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 33 1417 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 34 1431 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 38 1496 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 40 1523 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 42 1640 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 50 1666 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 51 1668 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 52 1695 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 65 1781 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 44 1649 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii   49A   1658.1 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii   49B   1658.2 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 56 1707 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 80 1893 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  5  693 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  8  749 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 10  839 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 14  865 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 16  888 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 29 1326 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 35 1440 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 41 1524 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 46 1652 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 47 1653 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 48 1657 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 57 1709 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 61 1727 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 63 1762 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 67 1806 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 75 1881 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 77 1886 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  1  649 46A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  2  653 46A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 39 1497 16A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 24 1198 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 28 1243 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 43 1648 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 62 1746 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii  4  689 15A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 68 1822 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 69   1823A 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 70   1823B 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 71 1826 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 72 1860 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 81 1924 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 82 1929 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 85 1966 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 11  841 3A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 32 1415 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 45 1651 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 54 1697 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 58 1712 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 60 1725 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 66 1802 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 76 1883 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 78 1891 24A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 79 1892 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 83 1947 24A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 84 1964 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 53 1696 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 36 1458 49A34G26C29T32 A30G28C24T32 A16G14C15T15 A. baumannii 59 1716 9A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii  9  805 30A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 18  967 39A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 30 1322 48A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 26 1218 50A33G26C29T33 A29G28C26T31 A16G14C15T15 A. sp. 13TU 15  875 A1A32G26C28T35 A28G28C24T34 A16G14C15T15 A. sp. 13TU 17  895 A1A32G26C28T35 A28G28C24T34 A16G14C15T15 A. sp. 3 12  853 B7 A29G26C27T39A26G32C21T35 A16G14C15T15 A. johnsonii 25 1202 NEW1 A32G28C26T35A29G29C22T34 A16G14C15T15 A. sp. 2082 87 2082 NEW2 A33G27C26T35A31G28C20T35 A16G14C15T15

TABLE 16A Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified with CodonAnalysis Primer Pairs Targeting the gyrA Gene PP No: 2852 PP No: 2853 PPNo: 2854 Species Ibis# Isolate ST gyrA gyrA gyrA A. baumannii 54 536 3A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 87 665 3A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 8 80 10 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 9 91 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 10 92 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 11 131 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 12 137 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 21 218 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 26 242 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 94 678 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 1 9 10 A25G23C21T32 A29G28C21T43 A17G13C13T21A. baumannii 2 13 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii3 19 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 4 24 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 5 36 10 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 6 39 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 13 139 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 15 165 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 16 170 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 17 186 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 20 202 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 22 221 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 24 234 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 25 239 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 33 370 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 34 389 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 19 201 14 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 27 257 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 29 301 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 31 354 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 36 422 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 37 424 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 38 434 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 39 473 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 40 482 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 44 512 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 45 516 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 47 522 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 48 526 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 50 528 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 52 531 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 53 533 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 56 542 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 59 550 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 62 556 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 64 557 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 70 588 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 73 603 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 74 605 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 75 606 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 77 611 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 79 622 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 83 643 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 85 653 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 89 669 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 93 674 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 23 228 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 32 369 52 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 35 393 52 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 30 339 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 41 485 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 42 493 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 43 502 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 46 520 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 49 527 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 51 529 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 65 562 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 68 579 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 57 546 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 58 548 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 60 552 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 61 555 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 63 557 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 66 570 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 67 578 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 69 584 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 71 593 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 72 602 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 76 609 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 78 621 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 80 625 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 81 628 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 82 632 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 84 649 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 86 655 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 88 668 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 90 671 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 91 672 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 92 673 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 18 196 55 A25G23C22T31 A29G28C21T43A17G13C13T21 A. baumannii 55 537 27 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 28 263 27 A25G23C22T31 A29G28C22T42A17G13C14T20 A. sp. 3 14 164 B7 A25G22C22T32 A30G29C22T40 A17G13C14T20mixture 7 71 — ND ND A17G13C15T19

TABLE 16B Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified with CodonAnalysis Primer Pairs Targeting the parC Gene PP No: 2846 PP No: 2847 PPNo: 2848 Species Ibis# Isolate ST parC parC parC A. baumannii 54 536 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 87 665 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 8 80 10 A33G26C28T34A29G28C25T32 A16G14C14T16 A. baumannii 9 91 10 A33G26C28T34 A29G28C25T32A16G14C14T16 A. baumannii 10 92 10 A33G26C28T34 A29G28C25T32 ND A.baumannii 11 131 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii12 137 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 21 218 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 26 242 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 94 678 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 1 9 10 A33G26C29T33A29G28C26T31 A16G14C15T15 A. baumannii 2 13 10 A33G26C29T33 A29G28C26T31A16G14C15T15 A. baumannii 3 19 10 A33G26C29T33 A29G28C26T31 A16G14C15T15A. baumannii 4 24 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii5 36 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 6 39 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 13 139 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 15 165 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 16 170 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 17 186 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 20 202 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 22 221 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 24 234 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 25 239 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 33 370 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 34 389 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 19 201 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 27 257 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 29 301 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 31 354 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 36 422 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 37 424 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 38 434 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 39 473 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 40 482 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 44 512 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 45 516 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 47 522 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 48 526 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 50 528 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 52 531 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 53 533 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 56 542 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 59 550 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 62 556 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 64 557 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 70 588 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 73 603 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 74 605 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 75 606 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 77 611 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 79 622 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 83 643 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 85 653 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 89 669 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 93 674 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 23 228 51A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 32 369 52A34G25C28T34 A30G27C25T32 A16G14C14T16 A. baumannii 35 393 52A34G25C28T34 A30G27C25T32 A16G14C14T16 A. baumannii 30 339 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 41 485 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 42 493 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 43 502 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 46 520 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 49 527 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 51 529 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 65 562 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 68 579 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 57 546 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 58 548 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 60 552 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 61 555 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 63 557 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 66 570 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 67 578 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 69 584 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 71 593 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 72 602 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 76 609 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 78 621 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 80 625 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 81 628 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 82 632 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 84 649 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 86 655 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 88 668 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 90 671 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 91 672 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 92 673 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 18 196 55A33G27C28T33 A29G28C25T31 A15G14C15T16 A. baumannii 55 537 27A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 28 263 27A33G26C29T33 A29G28C26T31 A16G14C15T15 A. sp. 3 14 164 B7 A35G25C29T32A30G28C17T39 A16G14C15T15 mixture 7 71 — ND ND A17G14C15T14

TABLE 17A Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with Speciating PrimerPair No. 2922 and Triangulation Genotyping Analysis Primer Pair Nos.1151 and 1156 PP No: 2922 PP No: 1151 PP No: 1156 Species Ibis# IsolateST efp trpE Adk A. baumannii 20 1082 1 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 13  854 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 22 1162 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 27 1230 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 31 1367 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 37 1459 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 55 1700 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 64 1777 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 73 1861 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 74 1877 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 86 1972 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  3  684 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  6  720 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  7  726 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 19 1079 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 21 1123 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 23 1188 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 33 1417 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 34 1431 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 38 1496 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 40 1523 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 42 1640 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 50 1666 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 51 1668 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 52 1695 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 65 1781 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 44 1649 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii   49A   1658.1 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii   49B   1658.2 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 56 1707 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 80 1893 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  5  693 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii  8  749 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 10  839 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 14  865 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 16  888 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 29 1326 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 35 1440 14 A44G35C25T43 ND A44G32C27T37 A.baumannii 41 1524 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii46 1652 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 47 165314 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 48 1657 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 57 1709 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 61 1727 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 63 1762 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 67 1806 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 75 1881 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 77 1886 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii  1  649 46A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii  2  653 46A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 39 1497 16A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 24 1198 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 28 1243 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 43 1648 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 62 1746 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii  4  689 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 68 1822 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 69   1823A 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 70   1823B 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 71 1826 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 72 1860 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 81 1924 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 82 1929 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 85 1966 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 11  841 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 32 1415 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 45 1651 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 54 1697 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 58 1712 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 60 1725 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 66 1802 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 76 1883 24 NDA43G36C20T43 A44G32C27T37 A. baumannii 78 1891 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 79 1892 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 83 1947 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 84 1964 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 53 1696 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 36 1458 49 A44G35C25T43A44G35C22T41 A44G32C27T37 A. baumannii 59 1716 9 A44G35C25T43A44G35C21T42 A44G32C26T38 A. baumannii  9  805 30 A44G35C25T43A44G35C19T44 A44G32C27T37 A. baumannii 18  967 39 A45G34C25T43A44G35C22T41 A44G32C26T38 A. baumannii 30 1322 48 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 26 1218 50 A44G35C25T43A44G35C21T42 A44G32C26T38 A. sp. 13TU 15  875 A1 A47G33C24T43A46G32C20T44 A44G33C27T36 A. sp. 13TU 17  895 A1 A47G33C24T43A46G32C20T44 A44G33C27T36 A. sp. 3 12  853 B7 A46G35C24T42 A42G34C20T46A43G33C24T40 A. johnsonii 25 1202 NEW1 A46G35C23T43 A42G35C21T44A43G33C23T41 A. sp. 2082 87 2082 NEW2 A46G36C22T43 A42G32C20T48A42G34C23T41

TABLE 17B Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with TriangulationGenotyping Analysis Primer Pair Nos. 1158 and 1160 and 1165 PP No: 1158PP No: 1160 PP No: 1165 Species Ibis# Isolate ST mutY mutY fumC A.baumannii 20 1082 1 A27G21C25T22 A32G35C29T33 A40G33C30T36 A. baumannii13  854 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 22 116210 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 27 1230 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 31 1367 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 37 1459 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 55 1700 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 64 1777 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 73 1861 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 74 1877 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 86 1972 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii  3  684 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii  6  720 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii  7  726 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 19 1079 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 21 1123 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 23 1188 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 33 1417 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 34 1431 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 38 1496 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 40 1523 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 42 1640 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 50 1666 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 51 1668 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 52 1695 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 65 1781 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 44 1649 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii   49A   1658.1 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii   49B   1658.2 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 56 1707 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 80 1893 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii  5  693 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii  8  749 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 10  839 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 14  865 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 16  888 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 29 1326 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 35 1440 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 41 1524 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 46 1652 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 47 1653 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 48 1657 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 57 1709 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 61 1727 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 63 1762 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 67 1806 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 75 1881 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 77 1886 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii  1  649 46A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii  2  653 46A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 39 1497 16A29G19C26T21 A31G35C29T34 A40G34C29T36 A. baumannii 24 1198 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 28 1243 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 43 1648 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 62 1746 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii  4  689 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 68 1822 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 69   1823A 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 70   1823B 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 71 1826 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 72 1860 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 81 1924 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 82 1929 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 85 1966 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 11  841 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 32 1415 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 45 1651 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 54 1697 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 58 1712 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 60 1725 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 66 1802 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 76 1883 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 78 1891 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 79 1892 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 83 1947 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 84 1964 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 53 1696 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 36 1458 49A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 59 1716 9A27G21C25T22 A32G35C28T34 A39G33C30T37 A. baumannii  9  805 30A27G21C25T22 A32G35C28T34 A39G33C30T37 A. baumannii 18  967 39A27G21C26T21 A32G35C28T34 A39G33C30T37 A. baumannii 30 1322 48A28G21C24T22 A32G35C29T33 A40G33C30T36 A. baumannii 26 1218 50A27G21C25T22 A31G36C28T34 A40G33C29T37 A. sp. 13TU 15  875 A1A27G21C25T22 A30G36C26T37 A41G34C28T36 A. sp. 13TU 17  895 A1A27G21C25T22 A30G36C26T37 A41G34C28T36 A. sp. 3 12  853 B7 A26G23C23T23A30G36C27T36 A39G37C26T37 A. johnsonii 25 1202 NEW1 A25G23C24T23A30G35C30T34 A38G37C26T38 A. sp. 2082 87 2082 NEW2 A26G22C24T23A31G35C28T35 A42G34C27T36

TABLE 17C Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with TriangulationGenotyping Analysis Primer Pair Nos. 1167 and 1170 and 1171 PP No: 1167PP No: 1170 PP No: 1171 Species Ibis# Isolate ST fumC fumC ppa A.baumannii 20 1082 1 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii13  854 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 22 116210 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 27 1230 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 31 1367 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 37 1459 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 55 1700 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 64 1777 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 73 1861 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 74 1877 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 86 1972 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  3  684 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  6  720 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  7  726 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 19 1079 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 21 1123 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 23 1188 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 33 1417 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 34 1431 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 38 1496 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 40 1523 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 42 1640 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 50 1666 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 51 1668 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 52 1695 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 65 1781 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 44 1649 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii   49A   1658.1 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii   49B   1658.2 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 56 1707 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 80 1893 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  5  693 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii  8  749 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 10  839 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 14  865 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 16  888 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 29 1326 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 35 1440 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 41 1524 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 46 1652 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 47 1653 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 48 1657 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 57 1709 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 61 1727 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 63 1762 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 67 1806 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 75 1881 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 77 1886 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii  1  649 46A41G35C32T39 A37G28C20T51 A35G37C32T45 A. baumannii  2  653 46A41G35C32T39 A37G28C20T51 A35G37C32T45 A. baumannii 39 1497 16A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 24 1198 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 28 1243 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 43 1648 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 62 1746 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii  4  689 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 68 1822 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 69   1823A 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 70   1823B 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 71 1826 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 72 1860 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 81 1924 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 82 1929 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 85 1966 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 11  841 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 32 1415 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 45 1651 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 54 1697 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 58 1712 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 60 1725 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 66 1802 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 76 1883 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 78 1891 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 79 1892 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 83 1947 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 84 1964 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 53 1696 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 36 1458 49A40G35C34T38 A39G26C22T49 A35G37C30T47 A. baumannii 59 1716 9A40G35C32T40 A38G27C20T51 A36G35C31T47 A. baumannii  9  805 30A40G35C32T40 A38G27C21T50 A35G36C29T49 A. baumannii 18  967 39A40G35C33T39 A38G27C20T51 A35G37C30T47 A. baumannii 30 1322 48A40G35C35T37 A38G27C21T50 A35G37C30T47 A. baumannii 26 1218 50A40G35C34T38 A38G27C21T50 A35G37C33T44 A. sp. 13TU 15  875 A1A41G39C31T36 A37G26C24T49 A34G38C31T46 A. sp. 13TU 17  895 A1A41G39C31T36 A37G26C24T49 A34G38C31T46 A. sp. 3 12  853 B7 A43G37C30T37A36G27C24T49 A34G37C31T47 A. johnsonii 25 1202 NEW1 A42G38C31T36A40G27C19T50 A35G37C32T45 A. sp. 2082 87 2082 NEW2 A43G37C32T35A37G26C21T52 A35G38C31T45

TABLE 18A Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified with SpeciatingPrimer Pair No. 2922 and Triangulation Genotyping Analysis Primer PairNos. 1151 and 1156 PP No: 2922 PP No: 1151 PP No: 1156 Species Ibis#Isolate ST efp trpE adk A. baumannii 54 536 3 A44G35C24T44 A44G35C22T41A44G32C26T38 A. baumannii 87 665 3 A44G35C24T44 A44G35C22T41A44G32C26T38 A. baumannii 8 80 10 A45G34C25T43 A44G35C21T42 A44G32C26T38A. baumannii 9 91 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii10 92 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 11 131 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 12 137 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 21 218 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 26 242 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 94 678 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 1 9 10 A45G34C25T43A44G35C21T42 A44G32C26T38 A. baumannii 2 13 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 3 19 10 A45G34C25T43 A44G35C21T42 A44G32C26T38A. baumannii 4 24 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii5 36 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 6 39 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 13 139 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 15 165 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 16 170 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 17 186 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 20 202 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 22 221 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 24 234 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 25 239 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 33 370 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 34 389 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 19 201 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 27 257 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 29 301 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 31 354 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 36 422 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 37 424 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 38 434 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 39 473 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 40 482 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 44 512 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 45 516 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 47 522 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 48 526 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 50 528 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 52 531 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 53 533 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 56 542 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 59 550 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 62 556 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 64 557 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 70 588 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 73 603 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 74 605 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 75 606 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 77 611 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 79 622 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 83 643 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 85 653 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 89 669 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 93 674 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 23 228 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 32 369 52A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 35 393 52A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 30 339 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 41 485 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 42 493 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 43 502 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 46 520 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 49 527 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 51 529 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 65 562 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 68 579 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 57 546 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 58 548 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 60 552 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 61 555 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 63 557 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 66 570 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 67 578 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 69 584 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 71 593 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 72 602 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 76 609 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 78 621 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 80 625 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 81 628 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 82 632 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 84 649 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 86 655 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 88 668 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 90 671 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 91 672 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 92 673 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 18 196 55A44G35C25T43 A44G35C20T43 A44G32C27T37 A. baumannii 55 537 27A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 28 263 27A44G35C25T43 A44G35C19T44 A44G32C27T37 A. sp. 3 14 164 B7 A46G35C24T42A42G34C20T46 A43G33C24T40 mixture 7 71 ? mixture ND ND

TABLE 18B Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified withTriangulation Genotyping Analysis Primer Pair Nos. 1158, 1160 and 1165PP No: 1158 PP No: 1160 PP No: 1165 Species Ibis# Isolate ST mutY mutYfumC A. baumannii 54 536 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A.baumannii 87 665 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 880 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 9 91 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 10 92 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 11 131 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 12 137 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 21 218 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 26 242 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 94 678 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 1 9 10 A27G21C26T21A32G35C28T34 A40G33C30T36 A. baumannii 2 13 10 A27G21C26T21 A32G35C28T34A40G33C30T36 A. baumannii 3 19 10 A27G21C26T21 A32G35C28T34 A40G33C30T36A. baumannii 4 24 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii5 36 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 6 39 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 13 139 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 15 165 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 16 170 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 17 186 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 20 202 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 22 221 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 24 234 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 25 239 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 33 370 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 34 389 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 19 201 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 27 257 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 29 301 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 31 354 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 36 422 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 37 424 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 38 434 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 39 473 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 40 482 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 44 512 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 45 516 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 47 522 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 48 526 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 50 528 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 52 531 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 53 533 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 56 542 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 59 550 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 62 556 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 64 557 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 70 588 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 73 603 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 74 605 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 75 606 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 77 611 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 79 622 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 83 643 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 85 653 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 89 669 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 93 674 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 23 228 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 32 369 52A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 35 393 52A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 30 339 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 41 485 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 42 493 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 43 502 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 46 520 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49 527 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 51 529 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 65 562 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 68 579 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 57 546 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 58 548 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 60 552 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 61 555 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 63 557 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 66 570 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 67 578 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 69 584 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 71 593 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 72 602 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 76 609 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 78 621 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 80 625 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 81 628 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 82 632 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 84 649 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 86 655 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 88 668 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 90 671 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 91 672 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 92 673 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 18 196 55A27G21C25T22 A31G36C27T35 A40G33C29T37 A. baumannii 55 537 27A27G21C25T22 A32G35C28T34 A40G33C30T36 A. baumannii 28 263 27A27G21C25T22 A32G35C28T34 A40G33C30T36 A. sp. 3 14 164 B7 A26G23C23T23A30G36C27T36 A39G37C26T37 mixture 7 71 ? ND ND ND

TABLE 18C Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified withTriangulation Genotyping Analysis Primer Pair Nos. 1167, 1170 and 1171PP No: 1167 PP No: 1170 PP No: 1171 Species Ibis# Isolate ST fumC fumCppa A. baumannii 54 536 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A.baumannii 87 665 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 880 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 9 91 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 10 92 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 11 131 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 12 137 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 21 218 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 26 242 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 94 678 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 1 9 10 A41G34C34T38A38G27C21T50 A35G37C33T44 A. baumannii 2 13 10 A41G34C34T38 A38G27C21T50A35G37C33T44 A. baumannii 3 19 10 A41G34C34T38 A38G27C21T50 A35G37C33T44A. baumannii 4 24 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii5 36 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 6 39 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 13 139 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 15 165 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 16 170 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 17 186 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 20 202 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 22 221 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 24 234 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 25 239 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 33 370 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 34 389 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 19 201 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 27 257 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 29 301 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 31 354 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 36 422 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 37 424 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 38 434 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 39 473 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 40 482 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 44 512 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 45 516 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 47 522 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 48 526 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 50 528 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 52 531 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 53 533 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 56 542 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 59 550 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 62 556 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 64 557 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 70 588 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 73 603 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 74 605 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 75 606 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 77 611 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 79 622 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 83 643 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 85 653 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 89 669 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 93 674 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 23 228 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 32 369 52A40G35C34T38 A38G27C21T50 A35G37C31T46 A. baumannii 35 393 52A40G35C34T38 A38G27C21T50 A35G37C31T46 A. baumannii 30 339 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 41 485 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 42 493 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 43 502 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 46 520 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 49 527 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 51 529 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 65 562 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 68 579 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 57 546 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 58 548 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 60 552 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 61 555 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 63 557 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 66 570 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 67 578 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 69 584 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 71 593 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 72 602 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 76 609 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 78 621 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 80 625 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 81 628 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 82 632 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 84 649 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 86 655 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 88 668 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 90 671 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 91 672 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 92 673 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 18 196 55A42G34C33T38 A38G27C20T51 A35G37C31T46 A. baumannii 55 537 27A40G35C33T39 A38G27C20T51 A35G37C33T44 A. baumannii 28 263 27A40G35C33T39 A38G27C20T51 A35G37C33T44 A. sp. 3 14 164 B7 A43G37C30T37A36G27C24T49 A34G37C31T47 mixture 7 71 — ND ND ND

Base composition analysis of the samples obtained from Walter Reedhospital indicated that a majority of the strain types identified werethe same strain types already characterized by the OIF study of Example12. This is not surprising since at least some patients from whichclinical samples were obtained in OIF were transferred to the WalterReed Hospital (WRAIR). Examples of these common strain types include:ST10, ST11, ST12, ST14, ST15, ST16 and ST46. A strong correlation wasnoted between these strain types and the presence of mutations in thegyrA and parC which confer quinolone drug resistance.

In contrast, the results of base composition analysis of samplesobtained from Northwestern Medical Center indicate the presence of 4major strain types: ST10, ST51, ST53 and ST54. All of these strain typeshave the gyrA quinolone resistance mutation and most also have the parCquinolone resistance mutation, with the exception of ST35. Thisobservation is consistent with the current understanding that the gyrAmutation generally appears before the parC mutation and suggests thatthe acquisition of these drug resistance mutations is rather recent andthat resistant isolates are taking over the wild-type isolates. Anotherinteresting observation was that a single isolate of ST3 (isolate 841)displays a triangulation genotyping analysis pattern similar to otherisolates of ST3, but the codon analysis amplification product basecompositions indicate that this isolate has not yet undergone thequinolone resistance mutations in gyrA and parC.

The six isolates that represent species other than Acinetobacterbaumannii in the samples obtained from the Walter Reed Hospital wereeach found to not carry the drug resistance mutations.

The results described above involved analysis of 183 samples using themethods and compositions of the present invention. Results were providedto collaborators at the Walter Reed hospital and Northwestern Medicalcenter within a week of obtaining samples. This example highlights therapid throughput characteristics of the analysis platform and theresolving power of triangulation genotyping analysis and codon analysisfor identification of and determination of drug resistance in bacteria.

Example 14 Identification of Drug Resistance Genes and Virulence Factorsin Staphylococcus aureus

An eight primer pair panel was designed for identification of drugresistance genes and virulence factors of Staphylococcus aureus and isshown in Table 19. The primer sequences are found in Table 2 and arecross-referenced by the primer pair numbers, primer pair names or SEQ IDNOs listed in Table 19.

TABLE 19 Primer Pairs for Identification of Drug Resistance Genes andVirulence Factors in Staphylococcus aureus Forward Reverse Primer PrimerPrimer Pair (SEQ ID (SEQ ID Target No. Forward Primer Name NO:) ReversePrimer Name NO:) Gene 879 MECA_Y14051_4507_4530_F 288MECA_Y14051_4555_4581_R 1269 mecA 2056 MECI-R_NC003923-41798- 698MECI-R_NC003923-41798- 1420 MecI-R 41609_33_60_F 41609_86_113_R 2081ERMA_NC002952-55890- 217 ERMA_NC002952-55890- 1167 ermA 56621_366_395_F56621_438_465_R 2086 ERMC_NC005908-2004- 399 ERMC_NC005908-2004- 1041ermC 2738_85_116_F 2738_173_206_R 2095 PVLUK_NC003923-1529595- 456PVLUK_NC003923-1529595- 1261 Pv-luk 1531285_688_713_F 1531285_775_804_R2249 TUFB_NC002758-615038- 430 TUFB_NC002758-615038- 1321 tufB616222_696_725_F 616222_793_820_R 2256 NUC_NC002758-894288- 174NUC_NC002758-894288- 853 Nuc 894974_316_345_F 894974_396_421_R 2313MUPR_X75439_2486_2516_F 172 MUPR_X75439_2548_2574_R 1360 mupR

Primer pair numbers 2256 and 2249 are confirmation primers designed withthe aim of high level identification of Staphylococcus aureus. The nucgene is a Staphylococcus aureus-specific marker gene. The tufB gene is auniversal housekeeping gene but the bioagent identifying amplicondefined by primer pair number 2249 provides a unique base composition(A43 G28 C19 T35) which distinguishes Staphylococcus aureus from othermembers of the genus Staphylococcus.

High level methicillin resistance in a given strain of Staphylococcusaureus is indicated by bioagent identifying amplicons defined by primerpair numbers 879 and 2056. Analyses have indicated that primer pairnumber 879 is not expected to prime S. sciuri homolog or Enterococcusfaecalis/faciem ampicillin-resistant PBP5 homologs.

Macrolide and erythromycin resistance in a given strain ofStaphylococcus aureus is indicated by bioagent identifying ampliconsdefined by primer pair numbers 2081 and 2086.

Resistance to mupriocin in a given strain of Staphylococcus aureus isindicated by bioagent identifying amplicons defined by primer pairnumber 2313.

Virulence in a given strain of Staphylococcus aureus is indicated bybioagent identifying amplicons defined by primer pair number 2095. Thisprimer pair can simultaneously and identify the pvl (lukS-PV) gene andthe lukD gene which encodes a homologous enterotoxin. A bioagentidentifying amplicon of the lukD gene has a six nucleobase lengthdifference relative to the lukS-PV gene.

A total of 32 blinded samples of different strains of Staphylococcusaureus were provided by the Center for Disease Control (CDC). Eachsample was analyzed by PCR amplification with the eight primer pairpanel, followed by purification and measurement of molecular masses ofthe amplification products by mass spectrometry. Base compositions forthe amplification products were calculated. The base compositionsprovide the information summarized above for each primer pair. Theresults are shown in Tables 20A and B. One result noted upon un-blindingof the samples is that each of the PVL+identifications agreed withPVL+identified in the same samples by standard PCR assays. These resultsindicate that the panel of eight primer pairs is useful foridentification of drug resistance and virulence sub-speciescharacteristics for Staphylococcus aureus. It is expected that a kitcomprising one or more of the members of this panel will be a usefulembodiment of the present invention.

TABLE 20A Drug Resistance and Virulence Identified in Blinded Samples ofVarious Strains of Staphylococcus aureus with Primer Pair Nos. 2081,2086, 2095 and 2256 Primer Primer Primer Sample Pair No. Pair No. PrimerPair No. Pair No. Index No. 2081 (ermA) 2086 (ermC) 2095 (pv-luk) 2256(nuc) CDC0010 − − PVL−/lukD+ + CDC0015 − − PVL+/lukD+ + CDC0019 − +PVL−/lukD+ + CDC0026 + − PVL−/lukD+ + CDC0030 + − PVL−/lukD+ + CDC004 −− PVL+/lukD+ + CDC0014 − + PVL+/lukD+ + CDC008 − − PVL−/lukD+ + CDC001 +− PVL−/lukD+ + CDC0022 + − PVL−/lukD+ + CDC006 + − PVL−/lukD+ + CDC007 −− PVL−/lukD+ + CDCVRSA1 + − PVL−/lukD+ + CDCVRSA2 + + PVL−/lukD+ +CDC0011 + − PVL−/lukD+ + CDC0012 − − PVL+/lukD− + CDC0021 + −PVL−/lukD+ + CDC0023 + − PVL−/lukD+ + CDC0025 + − PVL−/lukD+ + CDC005 −− PVL−/lukD+ + CDC0018 + − PVL+/lukD− + CDC002 − − PVL−/lukD+ +CDC0028 + − PVL−/lukD+ + CDC003 − − PVL−/lukD+ + CDC0013 − −PVL+/lukD+ + CDC0016 − − PVL−/lukD+ + CDC0027 + − PVL−/lukD+ + CDC0029 −− PVL+/lukD+ + CDC0020 − + PVL−/lukD+ + CDC0024 − − PVL−/lukD+ + CDC0031− − PVL−/lukD+ +

TABLE 20B Drug Resistance and Virulence Identified in Blinded Samples ofVarious Strains of Staphylococcus aureus with Primer Pair Nos. 2249,879, 2056, and 2313 Primer Primer Primer Pair No. Pair No. Pair No.Sample Primer Pair No. 2249 879 2056 2313 Index No. (tufB) (mecA)(mecI-R) (mupR) CDC0010 Staphylococcus aureus + + − CDC0015Staphylococcus aureus − − − CDC0019 Staphylococcus aureus + + − CDC0026Staphylococcus aureus + + − CDC0030 Staphylococcus aureus + + − CDC004Staphylococcus aureus + + − CDC0014 Staphylococcus aureus + + − CDC008Staphylococcus aureus + + − CDC001 Staphylococcus aureus + + − CDC0022Staphylococcus aureus + + − CDC006 Staphylococcus aureus + + + CDC007Staphylococcus aureus + + − CDCVRSA1 Staphylococcus aureus + + −CDCVRSA2 Staphylococcus aureus + + − CDC0011 Staphylococcus aureus − − −CDC0012 Staphylococcus aureus + + − CDC0021 Staphylococcus aureus + + −CDC0023 Staphylococcus aureus + + − CDC0025 Staphylococcus aureus + + −CDC005 Staphylococcus aureus + + − CDC0018 Staphylococcus aureus + + −CDC002 Staphylococcus aureus + + − CDC0028 Staphylococcus aureus + + −CDC003 Staphylococcus aureus + + − CDC0013 Staphylococcus aureus + + −CDC0016 Staphylococcus aureus + + − CDC0027 Staphylococcus aureus + + −CDC0029 Staphylococcus aureus + + − CDC0020 Staphylococcus aureus − − −CDC0024 Staphylococcus aureus + + − CDC0031 Staphylococcus scleiferi − −−

Example 15 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Staphylococcus aureus

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, a panelof eight triangulation genotyping analysis primer pairs was selected.The primer pairs are designed to produce bioagent identifying ampliconswithin six different housekeeping genes which are listed in Table 21.The primer sequences are found in Table 2 and are cross-referenced bythe primer pair numbers, primer pair names or SEQ ID NOs listed in Table21.

TABLE 21 Primer Pairs for Triangulation Genotyping Analysis ofStaphylococcus aureus Forward Reverse Primer Primer Primer Pair (SEQ ID(SEQ ID Target No. Forward Primer Name NO:) Reverse Primer Name NO:)Gene 2146 ARCC_NC003923-2725050- 437 ARCC_NC003923-2725050- 1137 arcC2724595_131_161_F 2724595_214_245_R 2149 AROE_NC003923-1674726- 530AROE_NC003923-1674726- 891 aroE 1674277_30_62_F 1674277_155_181_R 2150AROE_NC003923-1674726- 474 AROE_NC003923-1674726- 869 aroE1674277_204_232_F 1674277_308_335_R 2156 GMK_NC003923-1190906- 268GMK_NC003923-1190906- 1284 gmk 1191334_301_329_F 1191334_403_432_R 2157PTA_NC003923-628885- 418 PTA_NC003923-628885- 1301 pta 629355_237_263_F629355_314_345_R 2161 TPI_NC003923-830671- 318 TPI_NC003923-830671- 1300tpi 831072_1_34_F 831072_97_129_R 2163 YQI_NC003923-378916- 440YQI_NC003923-378916- 1076 yqi 379431_142_167_F 379431_259_284_R 2166YQI_NC003923-378916- 219 YQI_NC003923-378916- 1013 yqi 379431_275_300_F379431_364_396_R

The same samples analyzed for drug resistance and virulence in Example14 were subjected to triangulation genotyping analysis. The primer pairsof Table 21 were used to produce amplification products by PCR, whichwere subsequently purified and measured by mass spectrometry. Basecompositions were calculated from the molecular masses and are shown inTables 22A and 22B.

TABLE 22A Triangulation Genotyping Analysis of Blinded Samples ofVarious Strains of Staphylococcus aureus with Primer Pair Nos. 2146,2149, 2150 and 2156 Sample Primer Pair No. Primer Pair No. Primer PairNo. Primer Pair No. Index No. Strain 2146 (arcC) 2149(aroE) 2150 (aroE)2156 (gmk) CDC0010 COL A44 G24 C18 T29 A59 G24 C18 T51 A40 G36 C13 T43A50 G30 C20 T32 CDC0015 COL A44 G24 C18 T29 A59 G24 C18 T51 A40 G36 C13T43 A50 G30 C20 T32 CDC0019 COL A44 G24 C18 T29 A59 G24 C18 T51 A40 G36C13 T43 A50 G30 C20 T32 CDC0026 COL A44 G24 C18 T29 A59 G24 C18 T51 A40G36 C13 T43 A50 G30 C20 T32 CDC0030 COL A44 G24 C18 T29 A59 G24 C18 T51A40 G36 C13 T43 A50 G30 C20 T32 CDC004 COL A44 G24 C18 T29 A59 G24 C18T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0014 COL A44 G24 C18 T29 A59 G24C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC008 ???? A44 G24 C18 T29 A59G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC001 Mu50 A45 G23 C20 T27A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21 T31 CDC0022 Mu50 A45 G23 C20T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21 T31 CDC006 Mu50 A45 G23C20 T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21 T31 CDC0011 MRSA252A45 G24 C18 T28 A58 G24 C19 T51 A41 G36 C12 T43 A51 G29 C21 T31 CDC0012MRSA252 A45 G24 C18 T28 A58 G24 C19 T51 A41 G36 C12 T43 A51 G29 C21 T31CDC0021 MRSA252 A45 G24 C18 T28 A58 G24 C19 T51 A41 G36 C12 T43 A51 G29C21 T31 CDC0023 ST:110 A45 G24 C18 T28 A59 G24 C18 T51 A40 G36 C13 T43A50 G30 C20 T32 CDC0025 ST:110 A45 G24 C18 T28 A59 G24 C18 T51 A40 G36C13 T43 A50 G30 C20 T32 CDC005 ST:338 A44 G24 C18 T29 A59 G23 C19 T51A40 G36 C14 T42 A51 G29 C21 T31 CDC0018 ST:338 A44 G24 C18 T29 A59 G23C19 T51 A40 G36 C14 T42 A51 G29 C21 T31 CDC002 ST:108 A46 G23 C20 T26A58 G24 C19 T51 A42 G36 C12 T42 A51 G29 C20 T32 CDC0028 ST:108 A46 G23C20 T26 A58 G24 C19 T51 A42 G36 C12 T42 A51 G29 C20 T32 CDC003 ST:107A45 G23 C20 T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21 T31 CDC0013ST:12 ND A59 G24 C18 T51 A40 G36 C13 T43 A51 G29 C21 T31 CDC0016 ST:120A45 G23 C18 T29 A58 G24 C19 T51 A40 G37 C13 T42 A51 G29 C21 T31 CDC0027ST:105 A45 G23 C20 T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21 T31CDC0029 MSSA476 A45 G23 C20 T27 A58 G24 C19 T51 A40 G36 C13 T43 A50 G30C20 T32 CDC0020 ST:15 A44 G23 C21 T27 A59 G23 C18 T52 A40 G36 C13 T43A50 G30 C20 T32 CDC0024 ST:137 A45 G23 C20 T27 A57 G25 C19 T51 A40 G36C13 T43 A51 G29 C22 T30 CDC0031 *** No product No product No product Noproduct

TABLE 22B Triangulation Genotyping Analysis of Blinded Samples ofVarious Strains of Staphylococcus aureus with Primer Pair Nos. 2146,2149, 2150 and 2156 Sample Primer Pair No. Primer Pair No. Primer PairNo. Primer Pair No. Index No. Strain 2157 (pta) 2161 (tpi) 2163 (yqi)2166 (yqi) CDC0010 COL A32 G25 C23 T29 A51 G28 C22 T28 A41 G37 C22 T43A37 G30 C18 T37 CDC0015 COL A32 G25 C23 T29 A51 G28 C22 T28 A41 G37 C22T43 A37 G30 C18 T37 CDC0019 COL A32 G25 C23 T29 A51 G28 C22 T28 A41 G37C22 T43 A37 G30 C18 T37 CDC0026 COL A32 G25 C23 T29 A51 G28 C22 T28 A41G37 C22 T43 A37 G30 C18 T37 CDC0030 COL A32 G25 C23 T29 A51 G28 C22 T28A41 G37 C22 T43 A37 G30 C18 T37 CDC004 COL A32 G25 C23 T29 A51 G28 C22T28 A41 G37 C22 T43 A37 G30 C18 T37 CDC0014 COL A32 G25 C23 T29 A51 G28C22 T28 A41 G37 C22 T43 A37 G30 C18 T37 CDC008 unknown A32 G25 C23 T29A51 G28 C22 T28 A41 G37 C22 T43 A37 G30 C18 T37 CDC001 Mu50 A33 G25 C22T29 A50 G28 C22 T29 A42 G36 C22 T43 A36 G31 C19 T36 CDC0022 Mu50 A33 G25C22 T29 A50 G28 C22 T29 A42 G36 C22 T43 A36 G31 C19 T36 CDC006 Mu50 A33G25 C22 T29 A50 G28 C22 T29 A42 G36 C22 T43 A36 G31 C19 T36 CDC0011MRSA252 A32 G25 C23 T29 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30 C18 T37CDC0012 MRSA252 A32 G25 C23 T29 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30C18 T37 CDC0021 MRSA252 A32 G25 C23 T29 A50 G28 C22 T29 A42 G36 C22 T43A37 G30 C18 T37 CDC0023 ST:110 A32 G25 C23 T29 A51 G28 C22 T28 A41 G37C22 T43 A37 G30 C18 T37 CDC0025 ST:110 A32 G25 C23 T29 A51 G28 C22 T28A41 G37 C22 T43 A37 G30 C18 T37 CDC005 ST:338 A32 G25 C24 T28 A51 G27C21 T30 A42 G36 C22 T43 A37 G30 C18 T37 CDC0018 ST:338 A32 G25 C24 T28A51 G27 C21 T30 A42 G36 C22 T43 A37 G30 C18 T37 CDC002 ST:108 A33 G25C23 T28 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30 C18 T37 CDC0028 ST:108A33 G25 C23 T28 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30 C18 T37 CDC003ST:107 A32 G25 C23 T29 A51 G28 C22 T28 A41 G37 C22 T43 A37 G30 C18 T37CDC0013 ST:12 A32 G25 C23 T29 A51 G28 C22 T28 A42 G36 C22 T43 A37 G30C18 T37 CDC0016 ST:120 A32 G25 C24 T28 A50 G28 C21 T30 A42 G36 C22 T43A37 G30 C18 T37 CDC0027 ST:105 A33 G25 C22 T29 A50 G28 C22 T29 A43 G36C21 T43 A36 G31 C19 T36 CDC0029 MSSA476 A33 G25 C22 T29 A50 G28 C22 T29A42 G36 C22 T43 A36 G31 C19 T36 CDC0020 ST:15 A33 G25 C22 T29 A50 G28C21 T30 A42 G36 C22 T43 A36 G31 C18 T37 CDC0024 ST:137 A33 G25 C22 T29A51 G28 C22 T28 A42 G36 C22 T43 A37 G30 C18 T37 CDC0031 *** A34 G25 C25T25 A51 G27 C24 T27 No product No product Note: *** The sample CDC0031was identified as Staphylococcus scleiferi as indicated in Example 14.Thus, the triangulation genotyping primers designed for Staphylococcusaureus would generally not be expected to prime and produceamplification products of this organism. Tables 22A and 22B indicatethat amplification products are obtained for this organism only withprimer pair numbers 2157 and 2161.

A total of thirteen different genotypes of Staphylococcus aureus wereidentified according to the unique combinations of base compositionsacross the eight different bioagent identifying amplicons obtained withthe eight primer pairs. These results indicate that this eight primerpair panel is useful for analysis of unknown or newly emerging strainsof Staphylococcus aureus. It is expected that a kit comprising one ormore of the members of this panel will be a useful embodiment of thepresent invention.

Example 16 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Members of the Bacterial Genus Vibrio

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, a panelof eight triangulation genotyping analysis primer pairs was selected.The primer pairs are designed to produce bioagent identifying ampliconswithin seven different housekeeping genes which are listed in Table 23.The primer sequences are found in Table 2 and are cross-referenced bythe primer pair numbers, primer pair names or SEQ ID NOs listed in Table23.

TABLE 23 Primer Pairs for Triangulation Genotyping Analysis of Membersof the Bacterial Genus Vibrio Forward Reverse Primer Primer Primer Pair(SEQ ID (SEQ ID Target No. Forward Primer Name NO:) Reverse Primer NameNO:) Gene 1098 RNASEP_VBC_331_349_F 325 RNASEP_VBC_388_414_R 1163 RNAseP 2000 CTXB_NC002505_46_70_F 278 CTXB_NC002505_132_162_R 1039 ctxB 2001FUR_NC002505_87_113_F 465 FUR_NC002505_205_228_R 1037 fur 2011GYRB_NC002505_1161_1190_F 148 GYRB_NC002505_1255_1284_R 1172 gyrB 2012OMPU_NC002505_85_110_F 190 OMPU_NC002505_154_180_R 1254 ompU 2014OMPU_NC002505_431_455_F 266 OMPU_NC002505_544_567_R 1094 ompU 2323CTXA_NC002505-1568114- 508 CTXA_NC002505-1568114- 1297 ctxA1567341_122_149_F 1567341_186_214_R 2927 GAPA_NC002505_694_721_F 259GAPA_NC_002505_29_58_R 1060 gapA

A group of 50 bacterial isolates containing multiple strains of bothenvironmental and clinical isolates of Vibrio cholerae, 9 other Vibriospecies, and 3 species of Photobacteria were tested using this panel ofprimer pairs. Base compositions of amplification products obtained withthese 8 primer pairs were used to distinguish amongst various speciestested, including sub-species differentiation within Vibrio choleraeisolates. For instance, the non-O1/non-O139 isolates were clearlyresolved from the O1 and the O139 isolates, as were several of theenvironmental isolates of Vibrio cholerae from the clinical isolates.

It is expected that a kit comprising one or more of the members of thispanel will be a useful embodiment of the present invention.

Example 17 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Members of the Bacterial Genus Pseudomonas

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, a panelof twelve triangulation genotyping analysis primer pairs was selected.The primer pairs are designed to produce bioagent identifying ampliconswithin seven different housekeeping genes which are listed in Table 24.The primer sequences are found in Table 2 and are cross-referenced bythe primer pair numbers, primer pair names or SEQ ID NOs listed in Table24.

TABLE 24 Primer Pairs for Triangulation Genotyping Analysis of Membersof the Bacterial Genus Pseudomonas Forward Reverse Primer Primer PrimerPair (SEQ ID (SEQ ID Target No. Forward Primer Name NO:) Reverse PrimerName NO:) Gene 2949 ACS_NC002516-970624- 376 ACS_NC002516-970624- 1265acsA 971013_299_316_F 971013_364_383_R 2950 ARO_NC002516-26883- 267ARO_NC002516-26883- 1341 aroE 27380_4_26_F 27380_111_128_R 2951ARO_NC002516-26883- 705 ARO_NC002516-26883- 1056 aroE 27380_356_377_F27380_459_484_R 2954 GUA_NC002516-4226546- 710 GUA_NC002516-4226546-1259 guaA 4226174_155_178_F 4226174_265_287_R 2956 GUA_NC002516-4226546-374 GUA_NC002516-4226546- 1111 guaA 4226174_242_263_F 4226174_355_371_R2957 MUT_NC002516-5551158- 545 MUT_NC002516-5551158- 978 mutL5550717_5_26_F 5550717_99_116_R 2959 NUO_NC002516-2984589- 249NUO_NC002516-2984589- 1095 nuoD 2984954_8_26_F 2984954_97_117_R 2960NUO_NC002516-2984589- 195 NUO_NC002516-2984589- 1376 nuoD2984954_218_239_F 2984954_301_326_R 2961 PPS_NC002516-1915014- 311PPS_NC002516-1915014- 1014 pps 1915383_44_63_F 1915383_140_165_R 2962PPS_NC002516-1915014- 365 PPS_NC002516-1915014- 1052 pps1915383_240_258_F 1915383_341_360_R 2963 TRP_NC002516-671831- 527TRP_NC002516-671831- 1071 trpE 672273_24_42_F 672273_131_150_R 2964TRP_NC002516-671831- 490 TRP_NC002516-671831- 1182 trpE 672273_261_282_F672273_362_383_R

It is expected that a kit comprising one or more of the members of thispanel will be a useful embodiment of the present invention.

The present invention includes any combination of the various speciesand subgeneric groupings falling within the generic disclosure. Thisinvention therefore includes the generic description of the inventionwith a proviso or negative limitation removing any subject matter fromthe genus, regardless of whether or not the excised material isspecifically recited herein.

While in accordance with the patent statutes, description of the variousembodiments and examples have been provided, the scope of the inventionis not to be limited thereto or thereby. Modifications and alterationsof the present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.

Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims, rather than by the specific exampleswhich have been presented by way of example.

Each reference (including, but not limited to, journal articles, U.S.and non-U.S. patents, patent application publications, internationalpatent application publications, gene bank gi or accession numbers,internet web sites, and the like) cited in the present application isincorporated herein by reference in its entirety.

1. An oligonucleotide primer pair comprising a forward primer and areverse primer, wherein said forward primer comprises the sequence ofSEQ ID NO:318, and wherein said reverse primer comprises a sequence 95%identical to SEQ ID NO:1300.
 2. The oligonucleotide primer pair of claim1 wherein said forward primer is SEQ ID NO:
 318. 3. The oligonucleotideprimer pair of claim 1, wherein said reverse primer comprises thesequence of SEQ ID NO:1300.
 4. The oligonucleotide primer pair of claim1 wherein said reverse primer is SEQ ID NO:
 1300. 5. The oligonucleotideprimer pair of claim 1 wherein at least one of said forward primer andsaid reverse primer comprises at least one modified nucleobase.
 6. Theoligonucleotide primer pair of claim 5 wherein at least one of said atleast one modified nucleobase is a mass modified nucleobase.
 7. Theoligonucleotide primer pair of claim 6 wherein said mass modifiednucleobase is 5-Iodo-C.
 8. The composition of claim 6 wherein said massmodified nucleobase comprises a molecular mass modifying tag.
 9. Theoligonucleotide primer pair of claim 5 wherein at least one of said atleast one modified nucleobase is a universal nucleobase.
 10. Theoligonucleotide primer pair of claim 9 wherein said universal nucleobaseis inosine.
 11. The oligonucleotide primer pair of claim 1, wherein atleast one of said forward primer and said reverse primer comprises a Tresidue at its 5′ end.
 12. A kit for identifying, determining one ormore characteristics of, or detecting a Staphylococcus aureus bioagentcomprising the oligonucleotide primer pair of claim 1 and at least oneadditional primer pair designed to hybridize to a Staphylococcus aureusgene encoding arcC, aroE, gmk, pta, yqi, or a combination thereof. 13.The kit of claim 12 further comprising at least one other additionalprimer pair designed to hybridize to a Staphylococcus aureus geneencoding mecA, mecR1, pvluk, or a combination thereof.
 14. The kit ofclaim 12 wherein said at least one additional primer pair comprises SEQID NOs: 437:1232, SEQ ID NOs: 590:891, SEQ ID NOs: 474:869, SEQ ID NOs:268:1284, SEQ ID NOs: 418:1301, SEQ ID NOs: 440:1076, SEQ ID NOs:219:1013, or a combination thereof.
 15. The kit of claim 12 wherein eachprimer of said at least one additional primer pair has at least 70%sequence identity with one of SEQ ID NOs: 437, 1232, 590, 891, 474, 869,268, 1284, 418, 1301, 318, 1300, 440, 1076, 219 and 1013.